Method of nanopore sequencing of concatenated nucleic acids

ABSTRACT

The invention relates to a new method of characterising two or more target polynucleotides using a pore. The method involves sequentially attaching to a first polynucleotide one or more subsequent polynucleotides to form a concatenated polynucleotide.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational application number PCT/GB2017/051493, filed May 25, 2017,and claims the benefit of GB application number 1609221.5, filed May 25,2016, each of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to a new method of characterising two or moretarget polynucleotides using a pore.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap polynucleotide (e.g. DNAor RNA) sequencing and identification technologies across a wide rangeof applications. Existing technologies are slow and expensive mainlybecause they rely on amplification techniques to produce large volumesof polynucleotide and require a high quantity of specialist fluorescentchemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct,electrical biosensors for polymers and a variety of small molecules. Inparticular, recent focus has been given to nanopores as a potential DNAsequencing technology.

When a potential is applied across a nanopore, there is a change in thecurrent flow when an analyte, such as a nucleotide, resides transientlyin the barrel for a certain period of time. Nanopore detection of thenucleotide gives a current change of known signature and duration. Inthe strand sequencing method, a single polynucleotide strand is passedthrough the pore and the identities of the nucleotides are derived.Strand sequencing can involve the use of a molecular brake to controlthe movement of the polynucleotide through the pore.

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated that it is possible toimprove the efficiency of transmembrane pore-based characterisation oftwo or more target polynucleotides by sequentially attaching the targetpolynucleotides together to form a concatenated polynucleotide. If thebinding of a second target polynucleotide to a first polynucleotideoccurs selectively in the sense that binding only occurs as the firsttarget polynucleotide moves through the pore, the time taken for thesecond target polynucleotide to contact the pore is reduced and bindingof the target polynucleotides present in a sample in the absence of thepore is avoided. Concatenation of the target polynucleotides is onlyachieved when a first target polynucleotide moves through a pore. Thisis important because concatenation of the target polynucleotides insolution would result in a reduction of the number of free ends thatcould be captured by a pore.

Accordingly, the invention provides a method of characterising two ormore target polynucleotides. The method involves sequentially attachingto a first polynucleotide one or more subsequent polynucleotides to forma concatenated polynucleotide. The method comprises:

(a) contacting a first target polynucleotide with a transmembrane porein a membrane such that the first target polynucleotide moves throughthe pore;

(b) sequentially attaching to the first target polynucleotide one ormore subsequent target polynucleotides to provide a concatenatedpolynucleotide within which the target polynucleotides move through thepore in attachment order, wherein a subsequent target polynucleotide isselectively attached to the preceding target polynucleotide in theattachment order when the preceding target polynucleotide moves throughthe pore; and

(c) taking one or more measurements which are indicative of one or morecharacteristics of the concatenated polynucleotide as it moves withrespect to the pore.

Also provided is a population of two or more polynucleotide Y adaptorsfor characterising two or more double stranded target polynucleotides,wherein each adaptor comprises first and second parts which are capableof hybridising together and wherein each first part is initiallyprotected from hybridisaton to the second part.

In addition, a kit for characterising two or more double stranded targetpolynucleotides comprising a population of Y adaptors of the inventionand a population of hairpin loops is provided.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the attachment of a subsequent target polynucleotide(labelled B) to the first target polynucleotide (labelled A) to producea concatenated polynucleotide. The attachment point in the first targetpolynucleotide was revealed for attachment as the first polynucleotidemoved through the pore (labelled C).

FIG. 2 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase T4Dda-(E94C/F98W/C109A/C136A/A360C) (SEQ ID NO: 24 with mutationsE94C/F98W/C109A/C136A/A360C and then (ΔM1)G1G2 (where (ΔM1)G1G2=deletionof M1 and then addition G1 and G2)) controlled the translocation of theconcatenated polynucleotide through an MspA nanopore. The top traceshows the controlled translocation of the concatenated polynucleotideand the lower trace labelled 1 shows zoomed in region 1 of the toptrace.

FIG. 3 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase T4Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of theconcatenated polynucleotide through an MspA nanopore. The top traceshows the controlled translocation of the concatenated polynucleotideand the lower trace labelled 2 shows zoomed in region 2 of the toptrace.

FIG. 4 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase T4Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of theconcatenated polynucleotide through an MspA nanopore. The top traceshows the controlled translocation of the concatenated polynucleotideand the lower trace labelled 3 shows zoomed in region 3 of the toptrace.

FIG. 5 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase T4Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of theconcatenated polynucleotide through an MspA nanopore. The top traceshows the controlled translocation of the concatenated polynucleotideand the lower trace labelled 4 shows zoomed in region 4 of the toptrace.

FIG. 6 shows example current traces (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase T4Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of theconcatenated polynucleotide through an MspA nanopore. The top traceshows the controlled translocation of the concatenated polynucleotideand the lower trace labelled 5 shows zoomed in region 5 of the toptrace.

FIG. 7 shows the attachment of a subsequent target polynucleotide(labelled B) to the first target polynucleotide (labelled A) using clickchemistry (see click chemistry linkage labelled D) to produce aconcatenated polynucleotide. The attachment point in the first targetpolynucleotide was revealed for attachment as the first polynucleotidemoved through the pore (labelled C).

FIG. 8 shows an example current trace (y-axis label=Current (pA), x-axislabel=Time (s) for both traces) of when a helicase T4Dda-(E94C/F98W/C109A/C136A/A360C) controlled the translocation of theconcatenated polynucleotide through an MspA nanopore. The firstpolynucleotide had 5 subsequent target polynucleotides attached to itusing click chemistry. The regions of the current trace which correspondto either the template or complement region of the first polynucleotide(T1 and C1) and each subsequent polynucleotide (T2-6, C2-6) as theytranslocated through the nanopore are identified. Spacers in the leaderand the hairpins of the polynucleotides allow more current to flow andproduce a spike in current as they translocated through the nanopore.The spacers are highlighted with *(a-k) and mark transitions between thetemplate and complement regions of the first and subsequentpolynucleotides which were attached to form the target concatenatedpolynucleotide.

FIG. 9 shows the attachment of a subsequent target polynucleotide(labelled B) to the first target polynucleotide (labelled A) to producea concatenated polynucleotide. The attachment point in the first targetpolynucleotide was revealed for attachment as the first polynucleotidemoved through the pore (labelled C). In this example the firstpolynucleotide has one type of click chemistry reactive group (forexample azide reactive groups) at either end and the subsequentpolynucleotide has a different type of click chemistry reactive group(for example DBCO reactive groups) at either end. The enzyme used tocontrol the movement of the DNA through the nanopore is not shown onthis figure.

FIG. 10 shows how only template strands of target polynucleotides can becharacterised and concatenated, where the method of attachment used tojoin the polynucleotides together is click chemistry. The concatenationadapter complex contains a motor protein and a release protein. Thisadapter ligated to both ends of a target polynucleotide. Both proteinsare stalled on the ligated adapter complex until the adaptor ligated toa target polynucleotide is captured by the pore. Once a firstpolynucleotide has been captured, the blocking chemistry used to stallthe proteins is overcome by both proteins. The motor protein thencontrols the interaction of the first polynucleotide with the pore, asdescribed previously, and the release protein, which can translocatemore quickly than the motor protein, separates the strands to expose asequence (3′ hybridisation site) in the 3′ end of the adaptor linked tothe end of the target polynucleotide that is complementary to a 5′nucleic acid sequence (5′ hybridisation site) of the leader strand of anadapter complex that is ligated to a second target polynucleotide. Withthe 3′ hybridisation site revealed, the 5′ hybridisation site in thesecond target polynucleotide can then hybridise to the revealed 3′hybridisation site and covalent coupling of the 3′ end of the firstpolynucleotide to the 5′ of a the second polynucleotide can occur. Thisprocess then repeats for further concatenation of targetpolynucleotides.

It is to be understood that Figures are for the purpose of illustratingparticular embodiments of the invention only, and are not intended to belimiting.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encodingthe MS-B 1 mutant MspA monomer. This mutant lacks the signal sequenceand includes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of theMS-B 1 mutant of the MspA monomer. This mutant lacks the signal sequenceand includes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one monomer ofα-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19):7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one monomer of α-HL-NN.

SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the polynucleotide sequence encoding the Phi29 DNApolymerase.

SEQ ID NO: 9 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 10 shows the codon optimised polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 11 shows the amino acid sequence of exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 12 shows the codon optimised polynucleotide sequence derivedfrom the xthA gene from E. coli. It encodes the exonuclease III enzymefrom E. coli.

SEQ ID NO: 13 shows the amino acid sequence of the exonuclease IIIenzyme from E. coli. This enzyme performs distributive digestion of 5′monophosphate nucleosides from one strand of double stranded DNA (dsDNA)in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′overhang of approximately 4 nucleotides.

SEQ ID NO: 14 shows the codon optimised polynucleotide sequence derivedfrom the recJ gene from T. thermophiles. It encodes the RecJ enzyme fromT. thermophilus (TthRecJ-cd).

SEQ ID NO: 15 shows the amino acid sequence of the RecJ enzyme from T.thermophilus (TthRecJ-cd). This enzyme performs processive digestion of5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 16 shows the codon optimised polynucleotide sequence derivedfrom the bacteriophage lambda exo (redX) gene. It encodes thebacteriophage lambda exonuclease.

SEQ ID NO: 17 shows the amino acid sequence of the bacteriophage lambdaexonuclease. The sequence is one of three identical subunits thatassemble into a trimer. The enzyme performs highly processive digestionof nucleotides from one strand of dsDNA, in a 5′-3′ direction(www.neb.com/nebecomm/products/productM0262.asp). Enzyme initiation on astrand preferentially requires a 5′ overhang of approximately 4nucleotides with a 5′ phosphate.

SEQ ID NO: 18 shows the amino acid sequence of He1308 Mbu.

SEQ ID NO: 19 shows the amino acid sequence of He1308 Csy.

SEQ ID NO: 20 shows the amino acid sequence of He1308 Tga.

SEQ ID NO: 21 shows the amino acid sequence of He1308 Mhu.

SEQ ID NO: 22 shows the amino acid sequence of TraI Eco.

SEQ ID NO: 23 shows the amino acid sequence of XPD Mbu.

SEQ ID NO: 24 shows the amino acid sequence of Dda 1993.

SEQ ID NO: 25 shows the amino acid sequence of Trwc Cba.

SEQ ID NO: 26 shows the codon optimised polynucleotide sequence encodingthe wild-type CsgG monomer from Escherichia coli Str. K-12 substr.MC4100. This monomer lacks the signal sequence.

SEQ ID NO: 27 shows the amino acid sequence of the mature form of thewild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100.This monomer lacks the signal sequence. The abbreviation used for thisCsgG=CsgG-Eco.

SEQ ID NOs: 28 to 42 show the sequences used in the Examples.

It is to be understood that sequences are not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apolynucleotide” includes two or more polynucleotides, reference to “ananchor” refers to two or more anchors, reference to “a helicase”includes two or more helicases, and reference to “a transmembrane pore”includes two or more pores and the like.

In this specification, where different amino acids at a specificposition are separated by the symbol “/”, the / symbol “/” means “or”.For instance, P108R/K means P108R or P108K. In this specification wheredifferent positions or different substitutions are separated by thesymbol “/”, the “/” symbol means “and”. For example, E94/P108 means E94and P108 or E94D/P108K means E94D and P108K.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Method of the Invention

The methods devised by the inventors have various advantages. In knownmethods of polynucleotide characterization using a transmembranenanopore, the pore is open for some time after characterising onepolynucleotide before a subsequent polynucleotide contacts the pore. Thenew method significantly reduces the open pore time betweenpolynucleotides because the subsequent polynucleotide is being recruitedas the preceding polynucleotide is being characterised. In someembodiments, open pore time may be abolished, substantially abolished orminimised. Not only does a reduced open pore time mean that the pore isdoing more characterisation (processing more polynucleotides) than apore in a conventional method, but it also means that there is a reducedchance of pore blocking (the likelihood of the pore becoming blocked islow, or relatively low compared to in conventional methods). Poreblocking tends to occur when there is an open pore state. If apolynucleotide strand is in the pore then there is a reduced chance ofblocking occurring as the pore is already “occupied”. Therefore, if theopen pore state time is reduced the chance of blocking is also reduced.A reduced open pore also means that the concentration of polynucleotidesneeded for characterisation is also reduced.

Any number of target polynucleotides can be investigated orcharacterised using the invention. For instance, the method of theinvention may concern characterising two or more polynucleotides, suchas 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more,500 or more, 1,000 or more, 5,000 or more, 10,000 or more, 50,000 ormore 100,000 or more, 1,000,000 or more or 5,000,000 or more,polynucleotides. In particular, 2 or more, 5 or more, 10 or more, 20 ormore, 50 or more, 100 or more, 500 or more, 1,000 or more, 5,000 ormore, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more,1,000,000 or more or 5,000,000 or more subsequent target polynucleotidesare attached to the first target polynucleotide.

The two or more target polynucleotides may be different from oneanother. The two or more polynucleotides may be two or more instances ofthe same polynucleotide. This allows proof reading. The two or moretarget polynucleotides may be derived from the fragmentation of a longertarget polynucleotide.

Concatenated Polynucleotide

The method comprises sequentially attaching to a first targetpolynucleotide one or more subsequent target polynucleotides to providea concatenated polynucleotide. The concatenated polynucleotide comprisesat least two target polynucleotides attached together.

The target polynucleotides move through the pore in attachment order.For instance, the first target polynucleotide moves through the poreimmediately before the second target polynucleotide. The second targetpolynucleotide moves through the pore immediately before the thirdtarget polynucleotide and so on. In other words, the first targetpolynucleotide moves through the pore followed by the second targetpolynucleotide. The second target polynucleotide moves through the porefollowed by the third target polynucleotide and so on.

Attachment

As target polynucleotides move through a pore, a subsequent targetpolynucleotide is selectively attached to the preceding targetpolynucleotide in the attachment order when the preceding targetpolynucleotide moves through the pore. For instance, the second targetpolynucleotide is selectively attached to the first targetpolynucleotide when the first target polynucleotide moves through thepore. The third target polynucleotide is selectively attached to thesecond target polynucleotide when the second target polynucleotide movesthrough the pore and so on.

In other words, the target polynucleotides are not attached to oneanother until a first target polynucleotide interacts with the pore. Theinteraction of a first target polynucleotide with the pore facilitatesthe attachment of the first target polynucleotide to a second targetpolynucleotide. The interaction with the pore may result in aconformational change in the first polynucleotide, such as for exampledehybridisaton/separation of the two strands of a double stranded targetpolynucleotide, or removal of a protecting molecule to reveal or exposea site on the first polynucleotide that can interact with a site on thesecond polynucleotide. After the said site on the first polynucleotideis revealed or exposed, the first polynucleotide attaches to the secondpolynucleotide. The occurrence of the attachment only when the firstpolynucleotide interacts with the pore is referred to herein as“selective attachment”. This is distinct from “selective hybridisation”which is used herein to describe base pair binding between complementaryregions of a first polynucleotide and a second polynucleotide.

Thus, the attachment is selective in the sense that a subsequent targetpolynucleotide cannot attach to the preceding target polynucleotide inthe absence of the pore or until the preceding target polynucleotidemoves through the pore. For instance, the attachment is selective in thesense that the second target polynucleotide cannot attach to the firsttarget polynucleotide in the absence of the pore or until the firsttarget polynucleotide moves through the pore. The third targetpolynucleotide cannot attach to the second target polynucleotide in theabsence of the pore or until the second target polynucleotide movesthrough the pore and so on.

Selective attachment may be achieved in any way. A part of the precedingtarget polynucleotide (e.g. a site on a first polypeptide which binds toa site on a second polypeptide) is initially protected from attachmentto the subsequent target polynucleotide and is revealed for attachmentas the preceding target polynucleotide moves through the pore. Forinstance, a part of the first target polynucleotide is initiallyprotected from attachment to the second target polynucleotide and isrevealed for attachment as the first target polynucleotide moves throughthe pore. A part of the second target polynucleotide is initiallyprotected from attachment to the third target polynucleotide and isrevealed for attachment as the second target polynucleotide movesthrough the pore and so on.

The part may be protected in any way. The part is typically protected bya molecule which prevents attachment. Movement of the preceding targetpolynucleotide through the pore may remove the molecule and reveal thepart for attachment. Any molecule may be used. For instance, a moleculemay occlude one of the click reactive groups discussed below. This mightbe, for example, pyrene to stack with the DBCO. If the part comprisesNi-NTA groups (which can attach to polyhistidine, such as 6×His, in thesubsequent target polynucleotide), the part may be protected withpolyhistidine, such as 6×His, in the same target polynucleotide and viceversa, i.e. the part comprises polyhistidine, such as 6×His, and isprotected by Ni-NTA groups. If the part comprises cyclodextrin (whichcan attach to amantadine in the subsequent polynucleotide), the part maybe protected by amantadine in the same target polynucleotide or viceversa. The part and the protecting molecule may be present on oppositestrands of a double stranded target polynucleotide. Separation of thestrands by the pore may then separate the protecting molecule from thepart and reveal the part for attachment.

The part of the preceding target polynucleotide may be protected byhybridisaton to a protecting polynucleotide. The protectingpolynucleotide may be separated from the part as the targetpolynucleotide moves through the pore. The protecting polynucleotide mayform part of the target polynucleotide as discussed in more detailbelow. The protecting polynucleotide may be a separate polynucleotide.As discussed in more detail below, the protecting polynucleotide mayprotect the part from hybridisation to a part of the subsequent targetpolynucleotide. Alternatively, the protecting polynucleotide may preventthe action of a single strand ligase on the part. The presenceprotecting polynucleotide (forming a double stranded region with thepart) means the ligase cannot function. Release of the protectingpolynucleotide would reveal the part as a substrate for the ligase.

A part of the subsequent target polynucleotide preferably selectivelyhybridises to a part of the preceding polynucleotide, e.g. the siterevealed on the preceding target polynucleotide as a result of itsinteraction with the pore is a nucleotide sequence that is complementaryto a nucleotide sequence on the subsequent polynucleotide. For instance,a part of the second target polynucleotide preferably selectivelyhybridises to a part of the first polynucleotide. A part of the thirdtarget polynucleotide preferably selectively hybridises to a part of thesecond target polynucleotide and so on.

The part of the preceding target polynucleotide is preferably initiallyprotected from hybridisation to the part of the subsequent targetpolynucleotide and is revealed for hybridisation as the preceding targetpolynucleotide moves through the pore. For instance, the part of thesecond target polynucleotide is preferably initially protected fromhybridisation to the part of the first target polynucleotide and isrevealed for hybridisation as the first target polynucleotide movesthrough the pore. The part of the third target polynucleotide ispreferably initially protected from hybridisation to the part of thesecond target polynucleotide and is revealed for hybridisation as thesecond target polynucleotide moves through the pore and so on.

The part of the preceding target polynucleotide preferably specificallyhybridises to the part of the subsequent target polynucleotide. Theparts of the target polynucleotides specifically hybridise when theyhybridise with preferential or high affinity to each other but do notsubstantially hybridise, do not hybridise or hybridise with only lowaffinity to other polynucleotides or sequences. The part of thepreceding target polynucleotide specifically hybridises to the part ofthe subsequent target polynucleotide if it hybridises to the part of thesubsequent target polynucleotide with a melting temperature (T_(m)) thatis at least 2° C., such as at least 3° C., at least 4° C., at least 5°C., at least 6° C., at least 7° C., at least 8° C., at least 9° C. or atleast 10° C., greater than its T_(m) for other sequences. Morepreferably, the part of the preceding target polynucleotide hybridisesto the part of the subsequent target polynucleotide with a T_(m) that isat least 2° C., such as at least 3° C., at least 4° C., at least 5° C.,at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least10° C., at least 20° C., at least 30° C. or at least 40° C., greaterthan its T_(m) for other sequences. Preferably, the part of thepreceding target polynucleotide hybridises to the part of the subsequenttarget polynucleotide with a T_(m) that is at least 2° C., such as atleast 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7°C., at least 8° C., at least 9° C., at least 10° C., at least 20° C., atleast 30° C. or at least 40° C., greater than its T_(m) for a sequencewhich differs from the part of the subsequent target polynucleotide byone or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides.The part of the preceding target polynucleotide typically hybridises tothe part of the subsequent target polynucleotide with a T_(m) of atleast 90° C., such as at least 92° C. or at least 95° C. T_(m) can bemeasured experimentally using known techniques, including the use of DNAmicroarrays, or can be calculated using publicly available T_(m)calculators, such as those available over the internet.

Conditions that permit the hybridisation are well-known in the art (forexample, Sambrook et al., 2001, Molecular Cloning: a laboratory manual,3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocolsin Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-lnterscience, New York (1995)). Hybridisation can be carriedout under low stringency conditions, for example in the presence of abuffered solution of 30 to 35% formamide, 1 M NaCl and 1% SDS (sodiumdodecyl sulfate) at 37° C. followed by a 20 wash in from 1× (0.1650 MNa⁺) to 2× (0.33 M Na⁺) SSC (standard sodium citrate) at 50° C.Hybridisation can be carried out under moderate stringency conditions,for example in the presence of a buffer solution of 40 to 45% formamide,1 M NaCl, and 1% SDS at 37° C., followed by a wash in from 0.5× (0.0825M Na⁺) to 1× (0.1650 M Na⁺) SSC at 55° C. Hybridisation can be carriedout under high stringency conditions, for example in the presence of abuffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37° C., followedby a wash in 0.1× (0.0165 M Na⁺) SSC at 60° C.

The part of the preceding target polynucleotide is preferablysubstantially complementary to the part of the subsequent targetpolynucleotide. The part of the preceding target polynucleotide maytherefore have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches across aregion of 5, 10, 15, 20, 21, 22, 30, 40 or 50 nucleotides compared withthe part of the subsequent target polynucleotide. The part of thepreceding target polynucleotide is preferably complementary to the partof the subsequent target polynucleotide.

Each part is typically 50 nucleotides or fewer, such as 40 nucleotidesor fewer, 30 nucleotides or fewer, 20 nucleotides or fewer, 12nucleotides or fewer, 10 nucleotides or fewer or 5 nucleotides or fewer.Each part is typically at least 4 nucleotides in length, such as atleast 5 nucleotides, at least 10 nucleotides or at least 12 nucleotidesin length.

Each target polynucleotide may comprise the same parts, one whichselectively hybridises to a part of the preceding polynucleotide (whenit is the subsequent target polynucleotide) and one which selectivelyhybridises to a part of the subsequent polynucleotide (when it is thepreceding polynucleotide). In such embodiment, the two parts in eachtarget polynucleotide must be protected from one another so that they donot attach to each other and only attach to a different targetpolynucleotide. The parts may be protected from one another in any ofthe ways discussed above.

In one embodiment, the part of the preceding, e.g. first, polynucleotidethat is revealed for attachment to the subsequent, e.g. second,polynucleotide is at, or close to, the opposite end of the precedingpolynucleotide to the end that is fed into the pore. Where thepolynucleotide passes through the pore in a 5′ to 3′ direction, the partof the preceding polynucleotide that attaches to the subsequentpolynucleotide is at, or close to, the 3′ end of the precedingpolynucleotide. The part of the subsequent polynucleotide that attachesto the preceding polynucleotide is then at, or close to, the 5′ end ofthe subsequent polynucleotide. A part close to the end of apolynucleotide is typically within, for example, 1 to 30 bases of theend of the polynucleotide, such as within 2 to 25, 3 to 20, 4 to 15 or 5to 10 bases from the end of the polynucleotide.

In a preferred embodiment, the preceding target polynucleotide is doublestranded, the part (attachment site) of the preceding targetpolynucleotide is in one strand (a first strand) and is hybridised tothe other strand (a second strand), and the part (attachment site) isrevealed for hybridisation as the two (first and second) strandsseparate as the preceding target polynucleotide moves through the pore.In this embodiment, the other (second) strand of the targetpolynucleotide is the protecting molecule. If the second strand movesthrough the pore, the two strands separate and the part (attachment siteon the first strand) is revealed for hybridisation to the part(attachment site) on the subsequent target polynucleotide. A specificversion of this embodiment is shown in FIG. 1.

The one (first) strand in the preceding target polynucleotide preferablyforms a loop structure at its end. The part (attachment site) of thepreceding target polynucleotide which hybridises to the part (attachmentsite) of the subsequent target polynucleotide is preferably adjacent to,but not part of the loop structure. In such an embodiment, hybridisationof the part in the subsequent target polynucleotide to the part in thepreceding target polynucleotide elongates the loop. This embodimentresults in the preceding and subsequent target polynucleotides beingattached by a loop which may dehybridise and move through the pore. Aspecific version of this embodiment is shown in FIG. 1.

In one embodiment, both strands of the double stranded preceding targetpolynucleotide are preferably linked at one end by a hairpin loop.Hairpin loops are discussed in more detail below. If the two strands arelinked, the two strands separate, the part is revealed for hybridisationto the part on subsequent target polynucleotide, the hairpin movesthrough the pore and then the strand comprising the part attached to thesubsequent target polynucleotide moves through the pore.

The other (second) strand at the other end of the preceding targetpolynucleotide preferably comprises a leader sequence whichpreferentially threads into the pore. This ensures that the other(second) strand enters the pore and the part (attachment site) isrevealed for hybridisation as the two strands separate. Suitable leadersequences are discussed in more detail below.

The subsequent target polynucleotide is preferably attached to the one(first) strand at the other end of the preceding target polynucleotide.As the other (second) strand, which preferably comprises a leadersequence, moves through the pore, the part (attachment site) on the one(first) strand is preferably revealed for attachment or hybridisation tothe part (attachment site) on the subsequent target polynucleotide.

The subsequent target polynucleotide is preferably double stranded. Inone embodiment, the two strands are preferably linked at one end by ahairpin loop. The other end of the subsequent target polynucleotide fromthe hairpin loop is preferably selectively attached to the precedingtarget polynucleotide. In particular, the other end of the subsequenttarget polynucleotide from the hairpin loop preferably comprises aleader sequence which is capable of selectively attaching to thepreceding target polynucleotide. The free end of the leader sequence ispreferably capable of hybridizing to the part of the preceding targetpolynucleotide. The free end of the leader sequence is preferably thepart of the subsequent target polynucleotide which specificallyhybridises to the part of the preceding target polynucleotide. Asdiscussed above, the part of the preceding target polynucleotide ispreferably adjacent to a loop structure in the preceding targetpolynucleotide.

The end of the leader sequence may be hybridised to a bridgingpolynucleotide which forms an overhang which is capable of selectivelyattaching to the preceding target polynucleotide. The overhang ispreferably capable of hybridizing to the part (attachment site) of thepreceding target polynucleotide. The overhang is preferably the part ofthe subsequent target polynucleotide which specifically hybridises tothe part of the preceding target polynucleotide. In other words, theoverhang comprises or constitutes the attachment site in the subsequenttarget polynucleotide. Hybridisation of the overhang to the part(attachment site) in the preceding target polynucleotide positions theleader of the subsequent polynucleotide adjacent to the part (attachmentsite) in the preceding target polynucleotide and the two parts(attachment sites) may be attached to one another. The bridgingpolynucleotide and/or the overhang can be any length and formed from anyof the polynucleotides discussed below.

The second (subsequent) target polynucleotide is preferably selectivelycovalently attached to the first (preceding) target polynucleotide. Forinstance, in one embodiment, the part of the preceding targetpolynucleotide may hybridise with the part of the second targetpolynucleotide (e.g. where the attachment sites in the first and secondpolynucleotides are complementary to one another) and then the twopolynucleotides may be covalently attached. Any form of covalentattachment may be used.

The subsequent (second) target polynucleotide is preferably covalentlyattached to the preceding (first) target polynucleotide using a ligase,a topoisomerase or by click chemistry. The ligase, topoisomerase orclick chemistry results in the formation of one or more covalent bondsbetween the first and second target polynucleotides.

Any ligase may be used, such as T4 DNA ligase, E. coli DNA ligase, TaqDNA ligase, Tma DNA ligase and 9° N DNA ligase. The ligase is preferablyT3 DNA ligase. This commercially available, for instance from, NewEngland BioLabs® Inc.

Any topoisomerase may be used. Suitable topopisomerases include, but arenot limited to, Vaccinia DNA Topoisomerase or Human DNA Topoisomerase I.

Click chemistry is advantageous because it does not typically involvethe use of enzymes. Click chemistry is a term first introduced by Kolbet al. in 2001 to describe an expanding set of powerful, selective, andmodular building blocks that work reliably in both small- andlarge-scale applications (Kolb H C, Finn, M G, Sharpless K B, Clickchemistry: diverse chemical function from a few good reactions, Angew.Chem. Int. Ed. 40 (2001) 2004-2021). They have defined the set ofstringent criteria for click chemistry as follows: “The reaction must bemodular, wide in scope, give very high yields, generate only inoffensivebyproducts that can be removed by nonchromatographic methods, and bestereospecific (but not necessarily enantioselective). The requiredprocess characteristics include simple reaction conditions (ideally, theprocess should be insensitive to oxygen and water), readily availablestarting materials and reagents, the use of no solvent or a solvent thatis benign (such as water) or easily removed, and simple productisolation. Purification if required must be by nonchromatographicmethods, such as crystallization or distillation, and the product mustbe stable under physiological conditions”.

Suitable example of click chemistry include, but are not limited to, thefollowing:

-   -   (a) copper-free variant of the 1,3 dipolar cycloaddition        reaction, where an azide reacts with an alkyne under strain, for        example in a cyclooctane ring;    -   (b) the reaction of an oxygen nucleophile on one linker with an        epoxide or aziridine reactive moiety on the other; and    -   (c) the Staudinger ligation, where the alkyne moiety can be        replaced by an aryl phosphine, resulting in a specific reaction        with the azide to give an amide bond.

Preferably the click chemistry reaction is the Cu (I) catalysed 1,3dipolar cycloaddition reaction between an alkyne and an azide. In apreferred embodiment, the first group is an azide group and the secondgroup is an alkyne group. Nucleic acid bases have already beensynthesised incorporating azide and alkyne groups in preferred positions(for example Kocalka P, El-Sagheer A H, Brown T, Rapid and efficient DNAstrand cross-linking by click chemistry, Chembiochem. 2008.9(8):1280-5). Alkyne groups are available commercially from BerryAssociates (Michigan, USA) and azide groups are synthesised by ATDBio.

If the preceding and subsequent polynucleotides or part thereof aremodified to include groups that can form covalent bonds, the modifiednucleotides are preferably offset from one another by one nucleotide inorder to achieve the link. This follows the published work of Tom Brown(Kocalka et al. (2008) ChemBiochem 9 8 1280-1285).

Other preferred groups for use in the invention are shown in thefollowing Table 1.

TABLE 1 Some preferred groups capable of forming covalent bonds ReactsName with Structure 1,4-Bis[3-(2- pyridyldithio)propionamido]butaneThiols

1,11-bis- Maleimidotriethyleneglycol Thiols

3,3′-Dithiodipropionic acid di(N- hydroxysuccinimide ester) Primaryamines

Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) Primaryamines

4,4′-Diisothiocyanatostilbene-2,2′- disulfonic acid disodium saltPrimary amines

Bis[2-(4- azidosalicylamido)ethyl]disulfide Photo- activated, non-specific

3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester Thiols,primary amines

4-Maleimidobutyric acid N- hydroxysuccinimide ester Thiols, primaryamines

Iodoacetic acid N- hydroxysuccinimide ester Thiols, primary amines

S-Acetylthioglycolic acid N- hydroxysuccinimide ester Thiols, primaryamines

Azide-PEG-maleimide Thiols, alkyne

Alkyne-PEG-maleimide Thiols, azide

Copper free click chemistry can be used in the invention because of itsdesirable properties. For example, it is fast, clean and not poisonoustowards proteins. A good example of this is maleimide or iodoacetamidelinking with a cyclooctyne functional group (DIBO). However, othersuitable bio-orthogonal chemistries include, but are not limited to,Staudinger chemistry, hydrazine or hydrazide/aldehyde or ketone reagents(HyNic+4FB chemistry, including all Solulink™ reagents), Diels-Alderreagent pairs and boronic acid/salicyhydroxamate reagents.

Preferably the reactive groups are azide and hexynl groups such as3AzideN and 5′-hexynl-G.

Preferred pairs of non-covalent reactive groups include, but are notlimited to, (i) Ni-NTA and polyhistidine, such as 6×His, and (ii)cyclodextrin and adamantine.

Polynucleotide

The two more target polynucleotides may be any type of polynucleotide. Apolynucleotide, such as a nucleic acid, is a macromolecule comprisingtwo or more nucleotides. The polynucleotide or nucleic acid may compriseany combination of any nucleotides. The nucleotides can be naturallyoccurring or artificial. One or more nucleotides in the polynucleotidecan be oxidised or methylated. One or more nucleotides in thepolynucleotide may be damaged. For instance, the polynucleotide maycomprise a pyrimidine dimer. Such dimers are typically associated withdamage by ultraviolet light and are the primary cause of skin melanomas.One or more nucleotides in the polynucleotide may be modified, forinstance with a label or a tag. Suitable labels are described below. Thepolynucleotide may comprise one or more spacers.

A nucleotide typically contains a nucleobase, a sugar and at least onephosphate group. The nucleobase and sugar form a nucleoside.

The nucleobase is typically heterocyclic. Nucleobases include, but arenot limited to, purines and pyrimidines and more specifically adenine(A), guanine (G), thymine (T), uracil (U) and cytosine (C).

The sugar is typically a pentose sugar. Nucleotide sugars include, butare not limited to, ribose and deoxyribose. The sugar is preferably adeoxyribose.

The polynucleotide preferably comprises the following nucleosides:deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT),deoxyguanosine (dG) and deoxycytidine (dC).

The nucleotide is typically a ribonucleotide or deoxyribonucleotide. Thenucleotide typically contains a monophosphate, diphosphate ortriphosphate. The nucleotide may comprise more than three phosphates,such as 4 or 5 phosphates. Phosphates may be attached on the 5′ or 3′side of a nucleotide. Nucleotides include, but are not limited to,adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidinemonophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidinemonophosphate, 5-hydroxymethylcytidine monophosphate, cytidinemonophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclicguanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate(dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate(dCMP) and deoxymethylcytidine monophosphate. The nucleotides arepreferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMPand dUMP.

A nucleotide may be abasic (i.e. lack a nucleobase). A nucleotide mayalso lack a nucleobase and a sugar (i.e. is a C3 spacer).

The nucleotides in the polynucleotide may be attached to each other inany manner. The nucleotides are typically attached by their sugar andphosphate groups as in nucleic acids. The nucleotides may be connectedvia their nucleobases as in pyrimidine dimers.

The polynucleotide may be single stranded or double stranded. At least aportion of the polynucleotide is preferably double stranded.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The polynucleotide can comprise onestrand of RNA hybridised to one strand of DNA. The polynucleotide may beany synthetic nucleic acid known in the art, such as peptide nucleicacid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA),locked nucleic acid (LNA) or other synthetic polymers with nucleotideside chains. The PNA backbone is composed of repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backboneis composed of repeating glycol units linked by phosphodiester bonds.The TNA backbone is composed of repeating threose sugars linked togetherby phosphodiester bonds. LNA is formed from ribonucleotides as discussedabove having an extra bridge connecting the 2′ oxygen and 4′ carbon inthe ribose moiety.

The polynucleotide is most preferably ribonucleic nucleic acid (RNA) ordeoxyribonucleic acid (DNA).

The polynucleotide can be any length. For example, the polynucleotidecan be at least 10, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 400 or at least 500nucleotides or nucleotide pairs in length. The polynucleotide can be1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotidesor nucleotide pairs in length or 100000 or more nucleotides ornucleotide pairs in length.

Sample

The target polynucleotides may be present in any suitable sample. Thesample may be a biological sample. The invention may be carried out invitro using at least one sample obtained from or extracted from anyorganism or microorganism. The organism or microorganism is typicallyarchaeal, prokaryotic or eukaryotic and typically belongs to one of thefive kingdoms: plantae, animalia, fungi, monera and protista. Theinvention may be carried out in vitro on at least one sample obtainedfrom or extracted from any virus. The sample is preferably a fluidsample. The sample typically comprises a body fluid of the patient. Thesample may be urine, lymph, saliva, mucus or amniotic fluid but ispreferably blood, plasma or serum. Typically, the sample is human inorigin, but alternatively it may be from another mammal animal such asfrom commercially farmed animals such as horses, cattle, sheep, fish,chickens or pigs or may alternatively be pets such as cats or dogs.Alternatively, the sample may be of plant origin, such as a sampleobtained from a commercial crop, such as a cereal, legume, fruit orvegetable, for example wheat, barley, oats, canola, maize, soya, rice,rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans,lentils, sugar cane, cocoa, broccoli or cotton.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of non-biological samples includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample is typically processed prior to being used in the invention,for example by centrifugation or by passage through a membrane thatfilters out unwanted molecules or cells, such as red blood cells. Thesample may be measured immediately upon being taken. The sample may alsobe typically stored prior to assay, preferably below −70° C.

Membrane

The polynucleotides are contacted with a transmembrane pore in amembrane. Any membrane may be used in accordance with the invention.Suitable membranes are well-known in the art. The membrane is preferablyan amphiphilic layer. An amphiphilic layer is a layer formed fromamphiphilic molecules, such as phospholipids, which have bothhydrophilic and lipophilic properties. The amphiphilic molecules may besynthetic or naturally occurring. Non-naturally occurring amphiphilesand amphiphiles which form a monolayer are known in the art and include,for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009,25, 10447-10450). Block copolymers are polymeric materials in which twoor more monomer sub-units are polymerised together to create a singlepolymer chain. Block copolymers typically have properties that arecontributed by each monomer sub-unit. However, a block copolymer mayhave unique properties that polymers formed from the individualsub-units do not possess. Block copolymers can be engineered such thatone of the monomer sub-units is hydrophobic (i.e. lipophilic), whilstthe other sub-unit(s) are hydrophilic whilst in aqueous media. In thiscase, the block copolymer may possess amphiphilic properties and mayform a structure that mimics a biological membrane. The block copolymermay be a diblock (consisting of two monomer sub-units), but may also beconstructed from more than two monomer sub-units to form more complexarrangements that behave as amphiphiles. The copolymer may be atriblock, tetrablock or pentablock copolymer. The membrane is preferablya triblock copolymer membrane.

Archaebacterial bipolar tetraether lipids are naturally occurring lipidsthat are constructed such that the lipid forms a monolayer membrane.These lipids are generally found in extremophiles that survive in harshbiological environments, thermophiles, halophiles and acidophiles. Theirstability is believed to derive from the fused nature of the finalbilayer. It is straightforward to construct block copolymer materialsthat mimic these biological entities by creating a triblock polymer thathas the general motif hydrophilic-hydrophobic-hydrophilic. This materialmay form monomeric membranes that behave similarly to lipid bilayers andencompass a range of phase behaviours from vesicles through to laminarmembranes. Membranes formed from these triblock copolymers hold severaladvantages over biological lipid membranes. Because the triblockcopolymer is synthesised, the exact construction can be carefullycontrolled to provide the correct chain lengths and properties requiredto form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are notclassed as lipid sub-materials; for example a hydrophobic polymer may bemade from siloxane or other non-hydrocarbon based monomers. Thehydrophilic sub-section of block copolymer can also possess low proteinbinding properties, which allows the creation of a membrane that ishighly resistant when exposed to raw biological samples. This head groupunit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical andenvironmental stability compared with biological lipid membranes, forexample a much higher operational temperature or pH range. The syntheticnature of the block copolymers provides a platform to customise polymerbased membranes for a wide range of applications.

The membrane is most preferably one of the membranes disclosed inInternational Application No. PCT/GB2013/052766 (published as WO2014/064443) or PCT/GB2013/052767 (published as WO 2014/064444).

The amphiphilic molecules may be chemically-modified or functionalisedto facilitate coupling of the polynucleotide.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically planar. The amphiphilic layer may be curved. Theamphiphilic layer may be supported. The amphiphilic layer may beconcave. The amphiphilic layer may be suspended from raised pillars suchthat the peripheral region of the amphiphilic layer (which is attachedto the pillars) is higher than the amphiphilic layer region. This mayallow the microparticle to travel, move, slide or roll along themembrane as described above.

Amphiphilic membranes are typically naturally mobile, essentially actingas two dimensional fluids with lipid diffusion rates of approximately10⁻⁸ cm s-1. This means that the pore and coupled polynucleotide cantypically move within an amphiphilic membrane.

The membrane may be a lipid bilayer. Lipid bilayers are models of cellmembranes and serve as excellent platforms for a range of experimentalstudies. For example, lipid bilayers can be used for in vitroinvestigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitablemethods are disclosed in the Example. Lipid bilayers are commonly formedby the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972;69: 3561-3566), in which a lipid monolayer is carried on aqueoussolution/air interface past either side of an aperture which isperpendicular to that interface. The lipid is normally added to thesurface of an aqueous electrolyte solution by first dissolving it in anorganic solvent and then allowing a drop of the solvent to evaporate onthe surface of the aqueous solution on either side of the aperture. Oncethe organic solvent has evaporated, the solution/air interfaces oneither side of the aperture are physically moved up and down past theaperture until a bilayer is formed. Planar lipid bilayers may be formedacross an aperture in a membrane or across an opening into a recess.

The method of Montal & Mueller is popular because it is a cost-effectiveand relatively straightforward method of forming good quality lipidbilayers that are suitable for protein pore insertion. Other commonmethods of bilayer formation include tip-dipping, painting bilayers andpatch-clamping of liposome bilayers.

Tip-dipping bilayer formation entails touching the aperture surface (forexample, a pipette tip) onto the surface of a test solution that iscarrying a monolayer of lipid. Again, the lipid monolayer is firstgenerated at the solution/air interface by allowing a drop of lipiddissolved in organic solvent to evaporate at the solution surface. Thebilayer is then formed by the Langmuir-Schaefer process and requiresmechanical automation to move the aperture relative to the solutionsurface.

For painted bilayers, a drop of lipid dissolved in organic solvent isapplied directly to the aperture, which is submerged in an aqueous testsolution. The lipid solution is spread thinly over the aperture using apaintbrush or an equivalent. Thinning of the solvent results in theformation of a lipid bilayer. However, complete removal of the solventfrom the bilayer is difficult and consequently the bilayer formed bythis method is less stable and more prone to noise duringelectrochemical measurement.

Patch-clamping is commonly used in the study of biological cellmembranes. The cell membrane is clamped to the end of a pipette bysuction and a patch of the membrane becomes attached over the aperture.The method has been adapted for producing lipid bilayers by clampingliposomes which then burst to leave a lipid bilayer sealing over theaperture of the pipette. The method requires stable, giant andunilamellar liposomes and the fabrication of small apertures inmaterials having a glass surface.

Liposomes can be formed by sonication, extrusion or the Mozafari method(Colas et al. (2007) Micron 38:841-847).

In a preferred embodiment, the lipid bilayer is formed as described inInternational Application No. PCT/GB08/004127 (published as WO2009/077734). Advantageously in this method, the lipid bilayer is formedfrom dried lipids. In a most preferred embodiment, the lipid bilayer isformed across an opening as described in WO2009/077734(PCT/GB08/004127).

A lipid bilayer is formed from two opposing layers of lipids. The twolayers of lipids are arranged such that their hydrophobic tail groupsface towards each other to form a hydrophobic interior. The hydrophilichead groups of the lipids face outwards towards the aqueous environmenton each side of the bilayer. The bilayer may be present in a number oflipid phases including, but not limited to, the liquid disordered phase(fluid lamellar), liquid ordered phase, solid ordered phase (lamellargel phase, interdigitated gel phase) and planar bilayer crystals(lamellar sub-gel phase, lamellar crystalline phase).

Any lipid composition that forms a lipid bilayer may be used. The lipidcomposition is chosen such that a lipid bilayer having the requiredproperties, such as surface charge, ability to support membraneproteins, packing density or mechanical properties, is formed. The lipidcomposition can comprise one or more different lipids. For instance, thelipid composition can contain up to 100 lipids. The lipid compositionpreferably contains 1 to 10 lipids. The lipid composition may comprisenaturally-occurring lipids and/or artificial lipids.

The lipids typically comprise a head group, an interfacial moiety andtwo hydrophobic tail groups which may be the same or different. Suitablehead groups include, but are not limited to, neutral head groups, suchas diacylglycerides (DG) and ceramides (CM); zwitterionic head groups,such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) andsphingomyelin (SM); negatively charged head groups, such asphosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol(PI), phosphatic acid (PA) and cardiolipin (CA); and positively chargedheadgroups, such as trimethylammonium-Propane (TAP). Suitableinterfacial moieties include, but are not limited to,naturally-occurring interfacial moieties, such as glycerol-based orceramide-based moieties. Suitable hydrophobic tail groups include, butare not limited to, saturated hydrocarbon chains, such as lauric acid(n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmiticacid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic(n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid(cis-9-Octadecanoic); and branched hydrocarbon chains, such asphytanoyl. The length of the chain and the position and number of thedouble bonds in the unsaturated hydrocarbon chains can vary. The lengthof the chains and the position and number of the branches, such asmethyl groups, in the branched hydrocarbon chains can vary. Thehydrophobic tail groups can be linked to the interfacial moiety as anether or an ester. The lipids may be mycolic acid.

The lipids can also be chemically-modified. The head group or the tailgroup of the lipids may be chemically-modified. Suitable lipids whosehead groups have been chemically-modified include, but are not limitedto, PEG-modified lipids, such as1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-2000]; functionalised PEG Lipids, such as1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000]; and lipids modified for conjugation, such as1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl). Suitablelipids whose tail groups have been chemically-modified include, but arenot limited to, polymerisable lipids, such as1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinatedlipids, such as1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine;deuterated lipids, such as1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linkedlipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. Thelipids may be chemically-modified or functionalised to facilitatecoupling of the polynucleotide.

The amphiphilic layer, for example the lipid composition, typicallycomprises one or more additives that will affect the properties of thelayer. Suitable additives include, but are not limited to, fatty acids,such as palmitic acid, myristic acid and oleic acid; fatty alcohols,such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols,such as cholesterol, ergosterol, lanosterol, sitosterol andstigmasterol; lysophospholipids, such as1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides.

In another preferred embodiment, the membrane is a solid state layer.Solid state layers can be formed from both organic and inorganicmaterials including, but not limited to, microelectronic materials,insulating materials such as HfO₂, Si₃N₄, Al₂O₃, and SiO, organic andinorganic polymers such as polyamide, plastics such as Teflon® orelastomers such as two-component addition-cure silicone rubber, andglasses. The solid state layer may be by atomic layer deposition (ALD).The ALD solid state layer may comprise alternating layers of HfO₂ andAl₂O₃. The solid state layer may be formed from monatomic layers, suchas graphene, or layers that are only a few atoms thick. Suitablegraphene layers are disclosed in International Application No.PCT/US2008/010637 (published as WO 2009/035647). Yusko et al., NatureNanotechnology, 2011; 6: 253-260 and US Patent Application No.2013/0048499 describe the delivery of proteins to transmembrane pores insolid state layers without the use of microparticles. The method of theinvention may be used to improve the delivery in the methods disclosedin these documents.

The method is typically carried out using (i) an artificial amphiphiliclayer comprising a pore, (ii) an isolated, naturally-occurring lipidbilayer comprising a pore, or (iii) a cell having a pore insertedtherein. The method is typically carried out using an artificialamphiphilic layer, such as an artificial triblock copolymer layer. Thelayer may comprise other transmembrane and/or intramembrane proteins aswell as other molecules in addition to the pore. Suitable apparatus andconditions are discussed below. The method of the invention is typicallycarried out in vitro.

Transmembrane Pore

A transmembrane pore is a structure that crosses the membrane to somedegree.

Typically, a transmembrane pore comprises a first opening and a secondopening with a lumen extending between the first opening and the secondopening. The transmembrane pore permits hydrated ions driven by anapplied potential to flow across or within the membrane. Thetransmembrane pore typically crosses the entire membrane so thathydrated ions may flow from one side of the membrane to the other sideof the membrane. However, the transmembrane pore does not have to crossthe membrane. It may be closed at one end. For instance, the pore may bea well, gap, channel, trench or slit in the membrane along which or intowhich hydrated ions may flow.

Any transmembrane pore may be used in the invention. The pore may bebiological or artificial. Suitable pores include, but are not limitedto, protein pores, polynucleotide pores and solid state pores. The poremay be a DNA origami pore (Langecker et al., Science, 2012; 338:932-936).

The transmembrane pore is preferably a transmembrane protein pore. Atransmembrane protein pore is a polypeptide or a collection ofpolypeptides that permits hydrated ions, such as polynucleotide, to flowfrom one side of a membrane to the other side of the membrane. In thepresent invention, the transmembrane protein pore is capable of forminga pore that permits hydrated ions driven by an applied potential to flowfrom one side of the membrane to the other. The transmembrane proteinpore preferably permits polynucleotides to flow from one side of themembrane, such as a triblock copolymer membrane, to the other. Thetransmembrane protein pore allows a polynucleotide, such as DNA or RNA,to be moved through the pore.

The transmembrane protein pore may be a monomer or an oligomer. The poreis preferably made up of several repeating subunits, such as at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, or at least 16 subunits. Thepore is preferably a hexameric, heptameric, octameric or nonameric pore.The pore may be a homo-oligomer or a hetero-oligomer.

The transmembrane protein pore typically comprises a barrel or channelthrough which the ions may flow. The subunits of the pore typicallysurround a central axis and contribute strands to a transmembrane βbarrel or channel or a transmembrane α-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typicallycomprises amino acids that facilitate interaction with s, such asnucleotides, polynucleotides or nucleic acids. These amino acids arepreferably located near a constriction of the barrel or channel. Thetransmembrane protein pore typically comprises one or more positivelycharged amino acids, such as arginine, lysine or histidine, or aromaticamino acids, such as tyrosine or tryptophan. These amino acids typicallyfacilitate the interaction between the pore and nucleotides,polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention canbe derived from β-barrel pores or α-helix bundle pores. β-barrel porescomprise a barrel or channel that is formed from β-strands. Suitableβ-barrel pores include, but are not limited to, β-toxins, such asα-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria, such as Mycobacterium smegmatis porin(Msp), for example MspA, MspB, MspC or MspD, CsgG, outer membrane porinF (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase Aand Neisseria autotransporter lipoprotein (NalP) and other pores, suchas lysenin. α-helix bundle pores comprise a barrel or channel that isformed from α-helices. Suitable α-helix bundle pores include, but arenot limited to, inner membrane proteins and a outer membrane proteins,such as WZA and ClyA toxin. The transmembrane pore may be derived fromlysenin. Suitable pores derived from CsgG are disclosed in InternationalApplication No. PCT/EP2015/069965. Suitable pores derived from lyseninare disclosed in International Application No. PCT/GB2013/050667(published as WO 2013/153359). The transmembrane pore may be derivedfrom or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Spl andhaemolytic protein fragaceatoxin C (FraC). The wild type α-hemolysinpore is formed of 7 identical monomers or sub-units (i.e., it isheptameric). The sequence of one monomer or sub-unit of α-hemolysin-NNis shown in SEQ ID NO: 4.

The transmembrane protein pore is preferably derived from Msp, morepreferably from MspA. Such a pore will be oligomeric and typicallycomprises 7, 8, 9 or 10 monomers derived from Msp. The pore may be ahomo-oligomeric pore derived from Msp comprising identical monomers.Alternatively, the pore may be a hetero-oligomeric pore derived from Mspcomprising at least one monomer that differs from the others. Preferablythe pore is derived from MspA or a homolog or paralog thereof.

A monomer derived from Msp typically comprises the sequence shown in SEQID NO: 2 or a variant thereof. SEQ ID NO: 2 is the MS-(B1)8 mutant ofthe MspA monomer. It includes the following mutations: D90N, D91N, D93N,D118R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide thathas an amino acid sequence which varies from that of SEQ ID NO: 2 andwhich retains its ability to form a pore. The ability of a variant toform a pore can be assayed using any method known in the art. Forinstance, the variant may be inserted into an amphiphilic layer alongwith other appropriate subunits and its ability to oligomerise to form apore may be determined. Methods are known in the art for insertingsubunits into membranes, such as amphiphilic layers. For example,subunits may be suspended in a purified form in a solution containing atriblock copolymer membrane such that it diffuses to the membrane and isinserted by binding to the membrane and assembling into a functionalstate. Alternatively, subunits may be directly inserted into themembrane using the “pick and place” method described in M. A. Holden, H.Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Over the entire length of the amino acid sequence of SEQ ID NO: 2, avariant will preferably be at least 50% homologous to that sequencebased on amino acid similarity or identity. More preferably, the variantmay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid similarity oridentity to the amino acid sequence of SEQ ID NO: 2 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid similarity or identity over a stretch of 100 or more,for example 125, 150, 175 or 200 or more, contiguous amino acids (“hardhomology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p 38′7-395). ThePILEUP and BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). Similarity canbe measured using pairwise identity or by applying a scoring matrix suchas BLOSUM62 and converting to an equivalent identity. Since theyrepresent functional rather than evolved changes, deliberately mutatedpositions would be masked when determining homology. Similarity may bedetermined more sensitively by the application of position-specificscoring matrices using, for example, PSIBLAST on a comprehensivedatabase of protein sequences. A different scoring matrix could be usedthat reflect amino acid chemico-physical properties rather thanfrequency of substitution over evolutionary time scales (e.g. charge).

SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. The variant maycomprise any of the mutations in the MspB, C or D monomers compared withMspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 5 to 7.In particular, the variant may comprise the following substitutionpresent in MspB: A138P. The variant may comprise one or more of thefollowing substitutions present in MspC: A96G, N102E and A138P. Thevariant may comprise one or more of the following mutations present inMspD: Deletion of G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V,D91G, A96Q, N102D, S103T, V104I, S136K and G141A. The variant maycomprise combinations of one or more of the mutations and substitutionsfrom Msp B, C and D. The variant preferably comprises the mutation L88N.A variant of SEQ ID NO: 2 has the mutation L88N in addition to all themutations of MS-B1 and is called MS-(B2)8. The pore used in theinvention is preferably MS-(B2)8. The variant of SEQ ID NO: 2 preferablycomprises one or more of D56N, D56F, E59R, G75S, G77S, A96D and Q126R. Avariant of SEQ ID NO: 2 has the mutations G75S/G77S/L88N/Q126R inaddition to all the mutations of MS-B1 and is called MS-B2C. The poreused in the invention is preferably MS-(B2)8 or MS-(B2C)8. The variantof SEQ ID NO: 2 preferably comprises N93D. The variant more preferablycomprises the mutations G75S/G77S/L88N/N93D/Q126R.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid.

The transmembrane protein pore is preferably derived from CsgG, morepreferably from CsgG from E. coli Str. K-12 substr. MC4100. Such a porewill be oligomeric and typically comprises 7, 8, 9 or 10 monomersderived from CsgG. The pore may be a homo-oligomeric pore derived fromCsgG comprising identical monomers. Alternatively, the pore may be ahetero-oligomeric pore derived from CsgG comprising at least one monomerthat differs from the others.

A monomer derived from CsgG typically comprises the sequence shown inSEQ ID NO: 27 or a variant thereof. A variant of SEQ ID NO: 27 is apolypeptide that has an amino acid sequence which varies from that ofSEQ ID NO: 27 and which retains its ability to form a pore. The abilityof a variant to form a pore can be assayed using any method known in theart as discussed above.

Over the entire length of the amino acid sequence of any one of SEQ IDNO: 27, a variant will preferably be at least 50% homologous to thatsequence based on amino acid similarity or identity. More preferably,the variant may be at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% and morepreferably at least 95%, 97% or 99% homologous based on amino acidsimilarity or identity to the amino acid sequence of SEQ ID NO: 27 overthe entire sequence. There may be at least 80%, for example at least85%, 90% or 95%, amino acid similarity or identity over a stretch of 100or more, for example 125, 150, 175 or 200 or more, contiguous aminoacids (“hard homology”). Homology can be measured as discussed above.

The variant of SEQ ID NO: 27 may comprise any of the mutations disclosedin International Application No. PCT/GB2015/069965 (published as WO2016/034591). The variant of SEQ ID NO: 27 preferably comprises one ormore of the following (i) one or more mutations at the followingpositions (i.e. mutations at one or more of the following positions)N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 andR192, such as one or more mutations at the following positions (i.e.mutations at one or more of the following positions) N40, D43, E44, S54,S57, Q62, E101, E131 and T150 or N40, D43, E44, E101 and E131; (ii)mutations at Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56; (iii) Q42R orQ42K; (iv) K49R; (v) N102R, N102F, N102Y or N102W; (vi) D149N, D149Q orD149R; (vii) E185N, E185Q or E185R; (viii) D195N, D195Q or D195R; (ix)E201N, E201Q or E201R; (x) E203N, E203Q or E203R; and (xi) deletion ofone or more of the following positions F48, K49, P50, Y51, P52, A53,S54, N55, F56 and S57. The variant may comprise any combination of (i)to (xi). If the variant comprises any one of (i) and (iii) to (xi), itmay further comprise a mutation at one or more of Y51, N55 and F56, suchas at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.

Preferred variants of SEQ ID NO: 27 which form pores in which fewernucleotides contribute to the current as the polynucleotide movesthrough the pore comprise Y51A/F56A, Y51A/F56N, Y51I/F56A, Y51L/F56A,Y51T/F56A, Y51I/F56N, Y51L/F56N or Y51T/F56N or more preferablyY511/F56A, Y51L/F56A or Y51T/F56A.

Preferred variants of SEQ ID NO: 27 which form pores displaying anincreased range comprise mutations at the following positions:

Y51, F56, D149, E185, E201 and E203;

N55 and F56;

Y51 and F56;

Y51, N55 and F56; or

F56 and N102.

Preferred variants of SEQ ID NO: 27 which form pores displaying anincreased range comprise:

Y51N, F56A, D149N, E185R, E201N and E203N;

N55S and F56Q;

Y51A and F56A;

Y51A and F56N;

Y511 and F56A;

Y51L and F56A;

Y51T and F56A;

Y511 and F56N;

Y51L and F56N;

Y51T and F56N;

Y51T and F56Q;

Y51A, N55S and F56A;

Y51A, N55S and F56N;

Y51T, N55S and F56Q; or

F56Q and N102R.

Preferred variants of SEQ ID NO: 27 which form pores in which fewernucleotides contribute to the current as the polynucleotide movesthrough the pore comprise mutations at the following positions:

N55 and F56, such as N55X and F56Q, wherein X is any amino acid; or

Y51 and F56, such as Y51X and F56Q, wherein X is any amino acid.

Preferred variants of SEQ ID NO: 27 which form pores displaying anincreased throughput comprise mutations at the following positions:

D149, E185 and E203;

D149, E185, E201 and E203; or

D149, E185, D195, E201 and E203.

Preferred variants which form pores displaying an increased throughputcomprise:

D149N, E185N and E203N;

D149N, E185N, E201N and E203N;

D149N, E185R, D195N, E201N and E203N; or

D149N, E185R, D195N, E201R and E203N.

Preferred variants of SEQ ID NO: 7 which form pores in which capture ofthe polynucleotide is increased comprise the following mutations:

D43N/Y51T/F56Q;

E44N/Y51T/F56Q;

D43N/E44N/Y51T/F56Q;

Y51T/F56Q/Q62R;

D43N/Y51T/F56Q/Q62R;

E44N/Y51T/F56Q/Q62R; or

D43N/E44N/Y51T/F56Q/Q62R.

Preferred variants of SEQ ID NO: 27 comprise the following mutations:

D149R/E185R/E201R/E203R or Y51T/F56Q/D149R/E185R/E201R/E203R;

D149N/E185N/E201N/E203N or Y51T/F56Q/D149N/E185N/E201N/E203N;

E201R/E203R or Y51T/F56Q/E201R/E203R

E201N/E203R or Y51T/F56Q/E201N/E203R;

E203R or Y51T/F56Q/E203R;

E203N or Y51T/F56Q/E203N;

E201R or Y51T/F56Q/E201R;

E201N or Y51T/F56Q/E201N;

E185R or Y51T/F56Q/E185R;

E185N or Y51T/F56Q/E185N;

D149R or Y51T/F56Q/D149R;

D149N or Y51T/F56Q/D149N;

R142E or Y51T/F56Q/R142E;

R142N or Y51T/F56Q/R142N;

R192E or Y51T/F56Q/R192E; or

R192N or Y51T/F56Q/R192N.

Preferred variants of SEQ ID NO: 27 comprise the following mutations:

Y51A/F56Q/E101N/N102R;

Y51A/F56Q/R97N/N102G;

Y51A/F56Q/R97N/N102R;

Y51A/F56Q/R97N;

Y51A/F56Q/R97G;

Y51A/F56Q/R97L;

Y51A/F56Q/N102R;

Y51A/F56Q/N102F;

Y51A/F56Q/N102G;

Y51A/F56Q/E101R;

Y51A/F56Q/E101F;

Y51A/F56Q/E101N; or

Y51A/F56Q/E101G

The variant of SEQ ID NO: 27 may comprise any of the substitutionspresent in another CsgG homologue. Preferred CsgG homologues are shownin SEQ ID NOs: 3 to 7 and 26 to 41 of International Application No.PCT/GB2015/069965 (published as WO 2016/034591).

Any of the proteins described herein, such as the transmembrane proteinpores, may be modified to assist their identification or purification,for example by the addition of histidine residues (a his tag), asparticacid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag,a GST tag or a MBP tag, or by the addition of a signal sequence topromote their secretion from a cell where the polypeptide does notnaturally contain such a sequence. An alternative to introducing agenetic tag is to chemically react a tag onto a native or engineeredposition on the pore or construct. An example of this would be to reacta gel-shift reagent to a cysteine engineered on the outside of the pore.This has been demonstrated as a method for separating hemolysinhetero-oligomers (Chem Biol. 1997 July; 4(7):497-505).

The pore may be labelled with a revealing label. The revealing label maybe any suitable label which allows the pore to be detected. Suitablelabels include, but are not limited to, fluorescent molecules,radioisotopes e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

Any of the proteins described herein, such as the transmembrane proteinpores, may be made synthetically or by recombinant means. For example,the pore may be synthesised by in vitro translation and transcription(IVTT). The amino acid sequence of the pore may be modified to includenon-naturally occurring amino acids or to increase the stability of theprotein. When a protein is produced by synthetic means, such amino acidsmay be introduced during production. The pore may also be alteredfollowing either synthetic or recombinant production.

Any of the proteins described herein, such as the transmembrane proteinpores, can be produced using standard methods known in the art.Polynucleotide sequences encoding a pore or construct may be derived andreplicated using standard methods in the art. Polynucleotide sequencesencoding a pore or construct may be expressed in a bacterial host cellusing standard techniques in the art. The pore may be produced in a cellby in situ expression of the polypeptide from a recombinant expressionvector. The expression vector optionally carries an inducible promoterto control the expression of the polypeptide. These methods aredescribed in Sambrook, J. and Russell, D. (2001). Molecular Cloning: ALaboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

The pore may be produced in large scale following purification by anyprotein liquid chromatography system from protein producing organisms orafter recombinant expression. Typical protein liquid chromatographysystems include FPLC, AKTA systems, the Bio-Cad system, the Bio-RadBioLogic system and the Gilson HPLC system.

Microparticle

A microparticle may be used to deliver the target polynucleotide to thetransmembrane pore. Any number of microparticles can be used in themethod of the invention. For instance, the method of the invention mayuse a single microparticle or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50,100, 1,000, 5,000, 10,000, 100,000, 500,000 or 1,000,000 or moremicroparticles. If two or more microparticles are used, themicroparticles may be the same. Alternatively, a mixture of differentmicroparticles may be used.

Each microparticle may have one polynucleotide attached. Alternatively,each microparticle may have two or more polynucleotides, such as 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500 ormore, 1,000 or more, 5,000 or more, 10,000 or more, 100,000 or more,1000,000 or more or 5000,000 or more polynucleotides, attached. Amicroparticle may be substantially or completed coated or covered withpolynucleotide. A microparticle may have a polynucleotide attached oversubstantially all of or all of its surface. A microparticle may beattached to a polynucleotide via an adaptor. The adaptor may be aY-adaptor or a hairpin adaptor (see below)

A polynucleotide, i.e. a single instance of an polynucleotide, may beattached to two or more microparticles. A polynucleotide, i.e. a singleinstance of an polynucleotide, may be attached to any number of themicroparticles discussed above.

A microparticle is a microscopic particle whose size is typicallymeasured in micrometres (μm). Microparticles may also known asmicrospheres or microbeads. The microparticle may be a nanoparticle. Ananoparticle is a microscopic particle whose size is typically measuredin nanometres (nm).

A microparticle typically has a particle size of from about 0.001 μm toabout 500 μm. For instance, a nanoparticle may have a particle size offrom about 0.01 μm to about 200 μm or about 0.1 μm to about 100 μm. Moreoften, a microparticle has a particle size of from about 0.5 μm to about100 μm, or for instance from about 1 μm to about 50 μm. Themicroparticle may have a particle size of from about 1 nm to about 1000nm, such as from about 10 nm to about 500 nm, about 20 nm to about 200nm or from about 30 nm to about 100 nm.

A microparticle may be spherical or non-spherical. Sphericalmicroparticles may be called microspheres. Non-spherical particles mayfor instance be plate-shaped, needle-shaped, irregular or tubular. Theterm “particle size” as used herein means the diameter of the particleif the particle is spherical or, if the particle is non-spherical, thevolume-based particle size. The volume-based particle size is thediameter of the sphere that has the same volume as the non-sphericalparticle in question.

If two or more microparticles are used in the method, the averageparticle size of the microparticles may be any of the sizes discussedabove, such as from about 0.5 μm to about 500 μm. A population of two ormore microparticles preferably has a coefficient of variation (ratio ofthe standard deviation to the mean) of 10% or less, such as 5% or lessor 2% or less.

Any method may be used to determine the size of the microparticle.Suitable methods include, but are not limited to, flow cytometry (see,for example, Chandler et al., J Thromb Haemost. 2011 June;9(6):1216-24).

The microparticle may be formed from any material. The microparticle ispreferably formed from a ceramic, glass, silica, a polymer or a metal.The polymer may be a natural polymer, such as polyhydroxyalkanoate,dextran, polylactide, agarose, cellulose, starch or chitosan, or asynthetic polymer, such as polyurethane, polystyrene, poly(vinylchloride), silane or methacrylate. Suitable microparticles are known inthe art and are commercially available. Ceramic and glass microspheresare commercially available from 3M®. Silica and polymer microparticlesare commercially available from EPRUI Nanoparticles & Microspheres Co.Ltd. Microparticles are also commercially available from PolysciencesInc., Bangs Laboratories Inc. and Life Technologies.

The microparticle may be solid. The microparticle may be hollow. Themicroparticle may be formed from polymer fibers.

The microparticle may be derived from the kit used to extract andisolate the polynucleotide.

The surface of the microparticle may interact with and attach thepolynucleotide. The surface may naturally interact with thepolynucleotide without functionalisation. The surface of themicroparticle is typically functionalised to facilitate attachment ofthe polynucleotide. Suitable functionalisations are known in the art.For instance, the surface of the microparticle may be functionalisedwith a polyhistidine-tag (hexa histidine-tag, 6×His-tag, His6 tag orHis-tag®), Ni-NTA, streptavidin, biotin, an oligonucleotide, apolynucleotide (such as DNA, RNA, PNA, GNA, TNA or LNA), carboxylgroups, quaternary amine groups, thiol groups, azide groups, alkynegroups, DIBO, lipid, FLAG-tag (FLAG octapeptide, polynucleotide bindingproteins (including any of those discussed below), peptides, proteins,antibodies or antibody fragments. Antibody fragments are discussed inmore detail below. The microparticle may also be functionalised with anyof the linkers or groups discussed below with reference to attachment.

The microparticle may be functionalised with a molecule or group whichspecifically binds to the polynucleotide. In this instance, thepolynucleotide which will be attached to the microparticle and deliveredto the transmembrane pore may be called the target polynucleotide. Thisallows the microparticle to select or capture the target polynucleotidefrom a sample containing other polynucleotides. A molecule or groupspecifically binds to the target polynucleotide if it binds to thetarget polynucleotide with preferential or high affinity, but does notbind or binds with only low affinity to other or differentpolynucleotides. A molecule or group binds with preferential or highaffinity if it binds with a Kd of 1×10⁻⁶ M or less, more preferably1×10⁻⁷ M or less, 5×10⁻⁸M or less, more preferably 1×10⁻⁸M or less ormore preferably 5×10⁻⁹M or less. A molecule or group binds with lowaffinity if it binds with a Kd of 1×10⁻⁶ M or more, more preferably1×10⁻⁵ M or more, more preferably 1×10⁻⁴ M or more, more preferably1×10⁻³M or more, even more preferably 1×10⁻² M or more.

Preferably, the molecule or group binds to the target polynucleotidewith an affinity that is at least 10 times, such as at least 50, atleast 100, at least 200, at least 300, at least 400, at least 500, atleast 1000 or at least 10,000 times, greater than its affinity for otherpolynucleotides. Affinity can be measured using known binding assays,such as those that make use of fluorescence and radioisotopes.Competitive binding assays are also known in the art. The strength ofbinding between peptides or proteins and polynucleotides can be measuredusing nanopore force spectroscopy as described in Hornblower et al.,Nature Methods. 4: 315-317. (2007).

The microparticle may be functionalised with an oligonucleotide or apolynucleotide (such as any of those discussed above) which specificallyhybridises to the target polynucleotide or comprises a portion or regionwhich is complementary to a portion or region of the targetpolynucleotide. This allows the microparticle to select or capture thetarget polynucleotide from a sample containing other polynucleotides. Anoligonucleotide or polynucleotide specifically hybridises to a targetpolynucleotide when it hybridises with preferential or high affinity tothe target polynucleotide but does not substantially hybridise, does nothybridise or hybridises with only low affinity to other polynucleotide.An oligonucleotide or polynucleotide specifically hybridises if ithybridises to the target polynucleotide with a melting temperature(T_(m)) that is at least 2° C., such as at least 3° C., at least 4° C.,at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least9° C. or at least 10° C., greater than its T_(m) for other sequences.More preferably, the oligonucleotide or polynucleotide hybridises to thetarget polynucleotide with a T_(m) that is at least 2° C., such as atleast 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7°C., at least 8° C., at least 9° C., at least 10° C., at least 20° C., atleast 30° C. or at least 40° C., greater than its T_(m) for othernucleic acids. Preferably, the oligonucleotide or polynucleotidehybridises to the target polynucleotide with a T_(m) that is at least 2°C., such as at least 3° C., at least 4° C., at least 5° C., at least 6°C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., atleast 20° C., at least 30° C. or at least 40° C., greater than its T_(m)for a sequence which differs from the target polynucleotide by one ormore nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. Theoligonucleotide or polynucleotide typically hybridises to the targetpolynucleotide with a T_(m) of at least 90° C., such as at least 92° C.or at least 95° C. T_(m) can be measured experimentally using knowntechniques, including the use of DNA microarrays, or can be calculatedusing publicly available T_(m) calculators, such as those available overthe internet.

Conditions that permit the hybridisation are well-known in the art (forexample, Sambrook et al., 2001, Molecular Cloning: a laboratory manual,3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocolsin Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-lnterscience, New York (1995)). Hybridisation can be carriedout under low stringency conditions, for example in the presence of abuffered solution of 30 to 35% formamide, 1 M NaCl and 1% SDS (sodiumdodecyl sulfate) at 37° C. followed by a 20 wash in from 1× (0.1650 MNa⁺) to 2× (0.33 M Na⁺) SSC (standard sodium citrate) at 50° C.Hybridisation can be carried out under moderate stringency conditions,for example in the presence of a buffer solution of 40 to 45% formamide,1 M NaCl, and 1% SDS at 37° C., followed by a wash in from 0.5× (0.0825M Na⁺) to 1× (0.1650 M Na⁺) SSC at 55° C. Hybridisation can be carriedout under high stringency conditions, for example in the presence of abuffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37° C., followedby a wash in 0.1× (0.0165 M Na⁺) SSC at 60° C.

The oligonucleotide or polynucleotide may comprise a portion or regionwhich is substantially complementary to a portion or region of thetarget polynucleotide. The region or portion of the oligonucleotide orpolynucleotide may therefore have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moremismatches across a region of 5, 10, 15, 20, 21, 22, 30, 40 or 50nucleotides compared with the portion or region in the targetpolynucleotide.

A portion of region is typically 50 nucleotides or fewer, such as 40nucleotides or fewer, 30 nucleotides or fewer, 20 nucleotides or fewer,10 nucleotides or fewer or 5 nucleotides or fewer.

The microparticle is preferably paramagnetic or magnetic. Themicroparticle preferably comprises a paramagnetic or a superparamagneticmaterial or a paramagnetic or a superparamagnetic metal, such as iron.Any suitable magnetic microparticle may be used. For instance, magneticbeads commercially available from, for instance, Clontech, Promega,Invitrogen ThermoFisher Scientific and NEB, may be used. In someembodiments, the microparticle comprises a magnetic particle with anorganic group such as a metal-chelating group, such as nitrilotriaceticacid (NTA), attached. The organic component may, for instance, comprisea group selected from —C(═O)O—, —C—O—C—, —C(═O)—, —NH—, —C(═O)—NH,—C(═O)—CH₂—I, —S(═O)₂— and —S—. The organic component may comprise ametal chelating group, such as NTA (nitrilotriacetic acid). Usually, ametal such as gold, iron, nickel or cobalt is also attached to themetal-chelating group. Magnetic beads of this sort are commonly used forcapturing His-tagged proteins, but are also suitable for use in theinvention.

The microparticle is most preferably a His-Tag Dynabead® which iscommercially available from Life Technologies, Mag Strep beads from IBA,Streptavidin magnetic beads from NEB, Solid Phase ReversibleImmobilization (SPRI) beads or Agencourt AMPure XP beads from BeckmanCoulter or Dynabeads® MyOne™ Streptavidin C1 (ThermoFisher Scientific).

Coupling

The first target polynucleotide and/or the one or more subsequent targetpolynucleotides preferably comprise one or more anchors which arecapable of coupling to the membrane. The method preferably furthercomprises coupling the target polynucleotide to the membrane using theone or more anchors.

The anchor comprises a group which couples (or binds) to thepolynucleotide and a group which couples (or binds) to the membrane.Each anchor may covalently couple (or bind) to the polynucleotide and/orthe membrane.

The polynucleotide may be coupled to the membrane using any number ofanchors, such as 2, 3, 4 or more anchors. For instance, thepolynucleotide may be coupled to the membrane using two anchors each ofwhich separately couples (or binds) to both the polynucleotide andmembrane.

The one or more anchors may comprise one or more molecular brakes. Eachanchor may comprise one or more molecular brakes. The molecular brake(s)may be any of those discussed below.

If the membrane is an amphiphilic layer, such as a triblock copolymermembrane, the one or more anchors preferably comprise a polypeptideanchor present in the membrane and/or a hydrophobic anchor present inthe membrane. The hydrophobic anchor is preferably a lipid, fatty acid,sterol, carbon nanotube, polypeptide, protein or amino acid, for examplecholesterol, palmitate or tocopherol. In preferred embodiments, the oneor more anchors are not the pore.

The components of the membrane, such as the amphiphilic molecules,copolymer or lipids, may be chemically-modified or functionalised toform the one or more anchors. Examples of suitable chemicalmodifications and suitable ways of functionalising the components of themembrane are discussed in more detail below. Any proportion of themembrane components may be functionalised, for example at least 0.01%,at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or100%.

The polynucleotide may be coupled directly to the membrane. The one ormore anchors used to couple the polynucleotide to the membranepreferably comprise a linker. The one or more anchors may comprise oneor more, such as 2, 3, 4 or more, linkers. One linker may be used tocouple more than one, such as 2, 3, 4 or more, polynucleotides to themembrane.

Preferred linkers include, but are not limited to, polymers, such aspolynucleotides, polyethylene glycols (PEGs), polysaccharides andpolypeptides. These linkers may be linear, branched or circular. Forinstance, the linker may be a circular polynucleotide. Thepolynucleotide may hybridise to a complementary sequence on the circularpolynucleotide linker.

The one or more anchors or one or more linkers may comprise a componentthat can be cut or broken down, such as a restriction site or aphotolabile group.

Functionalised linkers and the ways in which they can couple moleculesare known in the art. For instance, linkers functionalised withmaleimide groups will react with and attach to cysteine residues inproteins. In the context of this invention, the protein may be presentin the membrane, may be the polynucleotide itself or may be used tocouple (or bind) to the polynucleotide. This is discussed in more detailbelow.

Crosslinkage of polynucleotides can be avoided using a “lock and key”arrangement.

Only one end of each linker may react together to form a longer linkerand the other ends of the linker each react with the polynucleotide ormembrane respectively. Such linkers are described in InternationalApplication No. PCT/GB10/000132 (published as WO 2010/086602).

The use of a linker is preferred in the sequencing embodiments discussedbelow. If a polynucleotide is permanently coupled directly to themembrane in the sense that it does not uncouple when interacting withthe pore, then some sequence data will be lost as the sequencing runcannot continue to the end of the polynucleotide due to the distancebetween the membrane and the pore. If a linker is used, then thepolynucleotide can be processed to completion.

The coupling may be permanent or stable. In other words, the couplingmay be such that the polynucleotide remains coupled to the membrane wheninteracting with the pore.

The coupling may be transient. In other words, the coupling may be suchthat the polynucleotide may decouple from the membrane when interactingwith the pore. For certain applications, such as aptamer detection andpolynucleotide sequencing, the transient nature of the coupling ispreferred. If a permanent or stable linker is attached directly toeither the 5′ or 3′ end of a polynucleotide and the linker is shorterthan the distance between the membrane and the transmembrane pore'schannel, then some sequence data will be lost as the sequencing runcannot continue to the end of the polynucleotide. If the coupling istransient, then when the coupled end randomly becomes free of themembrane, then the polynucleotide can be processed to completion.Chemical groups that form permanent/stable or transient links arediscussed in more detail below. The polynucleotide may be transientlycoupled to an amphiphilic layer or triblock copolymer membrane usingcholesterol or a fatty acyl chain. Any fatty acyl chain having a lengthof from 6 to 30 carbon atom, such as hexadecanoic acid, may be used.

In preferred embodiments, a polynucleotide, such as a nucleic acid, iscoupled to an amphiphilic layer such as a triblock copolymer membrane orlipid bilayer. Coupling of nucleic acids to synthetic lipid bilayers hasbeen carried out previously with various different tethering strategies.These are summarised in Table 2 below.

TABLE 2 Anchor Type of comprising coupling Reference Thiol StableYoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tetheredvesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7.Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior ofgiant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68Cholesterol Transient Pfeiffer, I. and F. Hook (2004). “Bivalentcholesterol-based coupling of oligonucletides to lipid membraneassemblies.” J Am Chem Soc 126(33): 10224-5 Surfactant Stable vanLengerich, B., R. J. Rawle, et al. (e.g. Lipid, “Covalent attachment oflipid vesicles to a Palmitate, fluid-supported bilayer allowsobservation of etc) DNA-mediated vesicle interactions.” Langmuir 26(11):8666-72

Synthetic polynucleotides and/or linkers may be functionalised using amodified phosphoramidite in the synthesis reaction, which is easilycompatible for the direct addition of suitable anchoring groups, such ascholesterol, tocopherol, palmitate, thiol, lipid and biotin groups.These different attachment chemistries give a suite of options forattachment to polynucleotides. Each different modification group couplesthe polynucleotide in a slightly different way and coupling is notalways permanent so giving different dwell times for the polynucleotideto the membrane. The advantages of transient coupling are discussedabove.

Coupling of polynucleotides to a linker or to a functionalised membranecan also be achieved by a number of other means provided that acomplementary reactive group or an anchoring group can be added to thepolynucleotide. The addition of reactive groups to either end of apolynucleotide has been reported previously. A thiol group can be addedto the 5′ of ssDNA or dsDNA using T4 polynucleotide kinase and ATPγS(Grant, G. P. and P. Z. Qin (2007). “A facile method for attachingnitroxide spin labels at the 5′ terminus of nucleic acids.” NucleicAcids Res 35(10): e77). An azide group can be added to the 5′-phosphateof ssDNA or dsDNA using T4 polynucleotide kinase andγ-[2-Azidoethyl]-ATP or γ-[6-Azidohexyl]-ATP. Using thiol or Clickchemistry a tether, containing either a thiol, iodoacetamide OPSS ormaleimide group (reactive to thiols) or a DIBO (dibenzocyclooxtyne) oralkyne group (reactive to azides), can be covalently attached to thepolynucleotide. A more diverse selection of chemical groups, such asbiotin, thiols and fluorophores, can be added using terminal transferaseto incorporate modified oligonucleotides to the 3′ of ssDNA (Kumar, A.,P. Tchen, et al. (1988). “Nonradioactive labeling of syntheticoligonucleotide probes with terminal deoxynucleotidyl transferase.” AnalBiochem 169(2): 376-82). Streptavidin/biotin and/orstreptavidin/desthiobiotin coupling may be used for any otherpolynucleotide. The Examples below describes how a polynucleotide can becoupled to a membrane using streptavidin/biotin andstreptavidin/desthiobiotin. It may also be possible that anchors may bedirectly added to polynucleotides using terminal transferase withsuitably modified nucleotides (e.g. cholesterol or palmitate).

The one or more anchors preferably couple the polynucleotide to themembrane via hybridisation. The hybridisation may be present in any partof the one or more anchors, such as between the one or more anchors andthe polynucleotide, within the one or more anchors or between the one ormore anchors and the membrane. Hybridisation in the one or more anchorsallows coupling in a transient manner as discussed above. For instance,a linker may comprise two or more polynucleotides, such as 3, 4 or 5polynucleotides, hybridised together. The one or more anchors mayhybridise to the polynucleotide. The one or more anchors may hybridisedirectly to the polynucleotide, directly to a Y adaptor and/or leadersequence attached to the polynucleotide or directly to a hairpin loopadaptor attached to the polynucleotide (as discussed in more detailbelow). Alternatively, the one or more anchors may be hybridised to oneor more, such as 2 or 3, intermediate polynucleotides (or “splints”)which are hybridised to the polynucleotide, to a Y adaptor and/or leadersequence attached to the polynucleotide or to a hairpin loop adaptorattached to the polynucleotide (as discussed in more detail below).

The one or more anchors may comprise a single stranded or doublestranded polynucleotide. One part of the anchor may be ligated to asingle stranded or double stranded polynucleotide analyte. Ligation ofshort pieces of ssDNA have been reported using T4 RNA ligase I (Troutt,A. B., M. G. McHeyzer-Williams, et al. (1992). “Ligation-anchored PCR: asimple amplification technique with single-sided specificity.” Proc NatlAcad Sci USA 89(20): 9823-5). Alternatively, either a single stranded ordouble stranded polynucleotide can be ligated to a double strandedpolynucleotide and then the two strands separated by thermal or chemicaldenaturation. To a double stranded polynucleotide, it is possible to addeither a piece of single stranded polynucleotide to one or both of theends of the duplex, or a double stranded polynucleotide to one or bothends. For addition of single stranded polynucleotides to the doublestranded polynucleotide, this can be achieved using T4 RNA ligase I asfor ligation to other regions of single stranded polynucleotides. Foraddition of double stranded polynucleotides to a double strandedpolynucleotide then ligation can be “blunt-ended”, with complementary 3′dA/dT tails on the polynucleotide and added polynucleotide respectively(as is routinely done for many sample prep applications to preventconcatemer or dimer formation) or using “sticky-ends” generated byrestriction digestion of the polynucleotide and ligation of compatibleadapters. Then, when the duplex is melted, each single strand will haveeither a 5′ or 3′ modification if a single stranded polynucleotide wasused for ligation or a modification at the 5′ end, the 3′ end or both ifa double stranded polynucleotide was used for ligation.

If the polynucleotide is a synthetic strand, the one or more anchors canbe incorporated during the chemical synthesis of the polynucleotide. Forinstance, the polynucleotide can be synthesised using a primer having areactive group attached to it.

Adenylated polynucleotides are intermediates in ligation reactions,where an adenosine-monophosphate is attached to the 5′-phosphate of thepolynucleotide. Various kits are available for generation of thisintermediate, such as the 5′ DNA Adenylation Kit from NEB. Bysubstituting ATP in the reaction for a modified nucleotide triphosphate,then addition of reactive groups (such as thiols, amines, biotin,azides, etc) to the 5′ of a polynucleotide can be possible. It may alsobe possible that anchors could be directly added to polynucleotidesusing a 5′ DNA adenylation kit with suitably modified nucleotides (e.g.cholesterol or palmitate).

A common technique for the amplification of sections of genomic DNA isusing polymerase chain reaction (PCR). Here, using two syntheticoligonucleotide primers, a number of copies of the same section of DNAcan be generated, where for each copy the 5′ of each strand in theduplex will be a synthetic polynucleotide. Single or multiplenucleotides can be added to 3′ end of single or double stranded DNA byemploying a polymerase. Examples of polymerases which could be usedinclude, but are not limited to, Terminal Transferase, Klenow and E.coli Poly(A) polymerase). By substituting ATP in the reaction for amodified nucleotide triphosphate then anchors, such as cholesterol,thiol, amine, azide, biotin or lipid, can be incorporated into doublestranded polynucleotides. Therefore, each copy of the amplifiedpolynucleotide will contain an anchor.

Ideally, the polynucleotide is coupled to the membrane without having tofunctionalise the polynucleotide. This can be achieved by coupling theone or more anchors, such as a molecular brake or a chemical group, tothe membrane and allowing the one or more anchors to interact with thepolynucleotide or by functionalizing the membrane. The one or moreanchors may be coupled to the membrane by any of the methods describedherein. In particular, the one or more anchors may comprise one or morelinkers, such as maleimide functionalised linkers.

In this embodiment, the polynucleotide is typically RNA, DNA, PNA, TNAor LNA and may be double or single stranded. This embodiment isparticularly suited to genomic DNA polynucleotides.

The one or more anchors can comprise any group that couples to, binds toor interacts with single or double stranded polynucleotides, specificnucleotide sequences within the polynucleotide or patterns of modifiednucleotides within the polynucleotide, or any other ligand that ispresent on the polynucleotide.

Suitable binding proteins for use in anchors include, but are notlimited to, E. coli single stranded binding protein, P5 single strandedbinding protein, T4 gp32 single stranded binding protein, the TOPO VdsDNA binding region, human histone proteins, E. coli HU DNA bindingprotein and other archaeal, prokaryotic or eukaryotic single stranded ordouble stranded polynucleotide (or nucleic acid) binding proteins,including those listed below.

The specific nucleotide sequences could be sequences recognised bytranscription factors, ribosomes, endonucleases, topoisomerases orreplication initiation factors. The patterns of modified nucleotidescould be patterns of methylation or damage.

The one or more anchors can comprise any group which couples to, bindsto, intercalates with or interacts with a polynucleotide. The group mayintercalate or interact with the polynucleotide via electrostatic,hydrogen bonding or Van der Waals interactions. Such groups include alysine monomer, poly-lysine (which will interact with ssDNA or dsDNA),ethidium bromide (which will intercalate with dsDNA), universal bases oruniversal nucleotides (which can hybridise with any polynucleotide) andosmium complexes (which can react to methylated bases). A polynucleotidemay therefore be coupled to the membrane using one or more universalnucleotides attached to the membrane. Each universal nucleotide may becoupled to the membrane using one or more linkers. The universalnucleotide preferably comprises one of the following nucleobases:hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, formylindole,3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole,5-nitroindazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring). Theuniversal nucleotide more preferably comprises one of the followingnucleosides: 2′-deoxyinosine, inosine, 7-deaza-2′-deoxyinosine,7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 2-O′-methylinosine,4-nitroindole 2′-deoxyribonucleoside, 4-nitroindole ribonucleoside,5-nitroindole 2′-deoxyribonucleoside, 5-nitroindole ribonucleoside,6-nitroindole 2′-deoxyribonucleoside, 6-nitroindole ribonucleoside,3-nitropyrrole 2′-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, anacyclic sugar analogue of hypoxanthine, nitroimidazole2′-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole2′-deoxyribonucleoside, 4-nitropyrazole ribonucleoside,4-nitrobenzimidazole 2′-deoxyribonucleoside, 4-nitrobenzimidazoleribonucleoside, 5-nitroindazole 2′-deoxyribonucleoside, 5-nitroindazoleribonucleoside, 4-aminobenzimidazole 2′-deoxyribonucleoside,4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside, phenylC-2′-deoxyribosyl nucleoside, 2′-deoxynebularine, 2′-deoxyisoguanosine,K-2′-deoxyribose, P-2′-deoxyribose and pyrrolidine. The universalnucleotide more preferably comprises 2′-deoxyinosine. The universalnucleotide is more preferably IMP or dIMP. The universal nucleotide ismost preferably dPMP (2′-Deoxy-P-nucleoside monophosphate) or dKMP(N6-methoxy-2,6-diaminopurine monophosphate).

The one or more anchors may couple to (or bind to) the polynucleotidevia Hoogsteen hydrogen bonds (where two nucleobases are held together byhydrogen bonds) or reversed Hoogsteen hydrogen bonds (where onenucleobase is rotated through 180° with respect to the othernucleobase). For instance, the one or more anchors may comprise one ormore nucleotides, one or more oligonucleotides or one or morepolynucleotides which form Hoogsteen hydrogen bonds or reversedHoogsteen hydrogen bonds with the polynucleotide. These types ofhydrogen bonds allow a third polynucleotide strand to wind around adouble stranded helix and form a triplex. The one or more anchors maycouple to (or bind to) a double stranded polynucleotide by forming atriplex with the double stranded duplex.

In this embodiment at least 1%, at least 10%, at least 25%, at least 50%or 100% of the membrane components may be functionalised.

Where the one or more anchors comprise a protein, they may be able toanchor directly into the membrane without further functonalisation, forexample if it already has an external hydrophobic region which iscompatible with the membrane. Examples of such proteins include, but arenot limited to, transmembrane proteins, intramembrane proteins andmembrane proteins. Alternatively the protein may be expressed with agenetically fused hydrophobic region which is compatible with themembrane. Such hydrophobic protein regions are known in the art.

The one or more anchors are preferably mixed with the polynucleotidebefore delivery to the membrane, but the one or more anchors may becontacted with the membrane and subsequently contacted with thepolynucleotide.

In another aspect the polynucleotide may be functionalised, usingmethods described above, so that it can be recognised by a specificbinding group. Specifically the polynucleotide may be functionalisedwith a ligand such as biotin (for binding to streptavidin), amylose (forbinding to maltose binding protein or a fusion protein), Ni-NTA (forbinding to poly-histidine or poly-histidine tagged proteins) or peptides(such as an antigen).

According to a preferred embodiment, the one or more anchors may be usedto couple a polynucleotide to the membrane when the polynucleotide isattached to a leader sequence which preferentially threads into thepore. Leader sequences are discussed in more detail below. Preferably,the polynucleotide is attached (such as ligated) to a leader sequencewhich preferentially threads into the pore. Such a leader sequence maycomprise a homopolymeric polynucleotide or an abasic region. The leadersequence is typically designed to hybridise to the one or more anchorseither directly or via one or more intermediate polynucleotides (orsplints).

In such instances, the one or more anchors typically comprise apolynucleotide sequence which is complementary to a sequence in theleader sequence or a sequence in the one or more intermediatepolynucleotides (or splints). In such instances, the one or more splintstypically comprise a polynucleotide sequence which is complementary to asequence in the leader sequence.

Any of the methods discussed above for coupling polynucleotides tomembranes, such as amphiphilic layers, can of course be applied to otherpolynucleotide and membrane combinations. In some embodiments, an aminoacid, peptide, polypeptide or protein is coupled to an amphiphiliclayer, such as a triblock copolymer layer or lipid bilayer. Variousmethodologies for the chemical attachment of such polynucleotides areavailable. An example of a molecule used in chemical attachment is EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Reactivegroups can also be added to the 5′ of polynucleotides using commerciallyavailable kits (Thermo Pierce, Part No. 22980). Suitable methodsinclude, but are not limited to, transient affinity attachment usinghistidine residues and Ni-NTA, as well as more robust covalentattachment by reactive cysteines, lysines or non natural amino acids.

Polynucleotide Characterisation

The method of the invention involves characterising the targetpolynucleotides. As the target polynucleotides are contacted with thepore, one or more measurements which are indicative of one or morecharacteristics of the target polynucleotides are taken as theconcatenated polynucleotide moves with respect to the pore.

The polynucleotides can be naturally occurring or artificial. Forinstance, the method may be used to verify the sequence of two or moremanufactured oligonucleotides. The methods are typically carried out invitro.

The method may involve measuring two, three, four or five or morecharacteristics of each polynucleotide. The one or more characteristicsare preferably selected from (i) the length of the polynucleotide, (ii)the identity of the polynucleotide, (iii) the sequence of thepolynucleotide, (iv) the secondary structure of the polynucleotide and(v) whether or not the polynucleotide is modified. Any combination of(i) to (v) may be measured in accordance with the invention, such as{i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii},{ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iv}, {i,ii,v},{i,iii,iv}, {i,iii,v}, {ii,iii,iv}, {ii,iii,v}, {ii,iv,v}, {iii,iv,v},{i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v}, {i,iii,iv,v}, {ii,iii,iv,v} or{i,ii,iii,iv,v}.

For (i), the length of the polynucleotide may be measured for example bydetermining the number of interactions between the polynucleotide andthe pore or the duration of interaction between the polynucleotide andthe pore.

For (ii), the identity of the polynucleotide may be measured in a numberof ways. The identity of the polynucleotide may be measured inconjunction with measurement of the sequence of the polynucleotide orwithout measurement of the sequence of the polynucleotide. The former isstraightforward; the polynucleotide is sequenced and thereby identified.The latter may be done in several ways. For instance, the presence of aparticular motif in the polynucleotide may be measured (withoutmeasuring the remaining sequence of the polynucleotide). Alternatively,the measurement of a particular electrical and/or optical signal in themethod may identify the polynucleotide as coming from a particularsource.

For (iii), the sequence of the polynucleotide can be determined asdescribed previously. Suitable sequencing methods, particularly thoseusing electrical measurements, are described in Stoddart D et al., ProcNatl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc.2010; 132(50):17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways.For instance, if the method involves an electrical measurement, thesecondary structure may be measured using a change in dwell time or achange in current flowing through the pore. This allows regions ofsingle-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured.The method preferably comprises determining whether or not thepolynucleotide is modified by methylation, by oxidation, by damage, withone or more proteins or with one or more labels, tags or spacers.Specific modifications will result in specific interactions with thepore which can be measured using the methods described below. Forinstance, methylcyotsine may be distinguished from cytosine on the basisof the current flowing through the pore during its interaction with eachnucleotide.

The methods may be carried out using any apparatus that is suitable forinvestigating a membrane/pore system in which a pore is present in amembrane. The method may be carried out using any apparatus that issuitable for transmembrane pore sensing. For example, the apparatuscomprises a chamber comprising an aqueous solution and a barrier thatseparates the chamber into two sections. The barrier typically has anaperture in which the membrane containing the pore is formed.Alternatively the barrier forms the membrane in which the pore ispresent.

The methods may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562 (WO 2008/102120).

A variety of different types of measurements may be made. This includeswithout limitation: electrical measurements and optical measurements. Asuitable optical method involving the measurement of fluorescence isdisclosed by J. Am. Chem. Soc. 2009, 131 1652-1653. Possible electricalmeasurements include: current measurements, impedance measurements,tunnelling measurements (Ivanov A P et al., Nano Lett. 2011 Jan. 12;11(1):279-85), and FET measurements (International Application WO2005/124888). Optical measurements may be combined with electricalmeasurements (Soni G V et al., Rev Sci Instrum. 2010 January;81(1):014301). The measurement may be a transmembrane currentmeasurement such as measurement of ionic current flowing through thepore.

Electrical measurements may be made using standard single channelrecording equipment as describe in Stoddart D et al., Proc Natl AcadSci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010;132(50):17961-72, and International Application WO 2000/28312.Alternatively, electrical measurements may be made using a multi-channelsystem, for example as described in International Application WO2009/077734 and International Application WO 2011/067559.

The method is preferably carried out with a potential applied across themembrane. The applied potential may be a voltage potential.Alternatively, the applied potential may be a chemical potential. Anexample of this is using a salt gradient across a membrane, such as anamphiphilic layer. A salt gradient is disclosed in Holden et al., J AmChem Soc. 2007 Jul. 11; 129(27):8650-5. In some instances, the currentpassing through the pore as a polynucleotide moves with respect to thepore is used to estimate or determine the sequence of thepolynucleotide. This is strand sequencing.

The methods may involve measuring the current passing through the poreas the polynucleotide moves with respect to the pore. Therefore theapparatus may also comprise an electrical circuit capable of applying apotential and measuring an electrical signal across the membrane andpore. The methods may be carried out using a patch clamp or a voltageclamp. The methods preferably involve the use of a voltage clamp.

The methods of the invention may involve the measuring of a currentpassing through the pore as the polynucleotide moves with respect to thepore. Suitable conditions for measuring ionic currents throughtransmembrane protein pores are known in the art and disclosed in theExample. The method is typically carried out with a voltage appliedacross the membrane and pore. The voltage used is typically from +5 V to−5 V, such as from +4 V to −4 V, +3 V to −3 V or +2 V to −2 V. Thevoltage used is typically from −600 mV to +600 mV or −400 mV to +400 mV.The voltage used is preferably in a range having a lower limit selectedfrom −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0mV and an upper limit independently selected from +10 mV, +20 mV, +50mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used ismore preferably in the range 100 mV to 240 mV and most preferably in therange of 120 mV to 220 mV. It is possible to increase discriminationbetween different nucleotides by a pore by using an increased appliedpotential.

The methods are typically carried out in the presence of any chargecarriers, such as metal salts, for example alkali metal salt, halidesalts, for example chloride salts, such as alkali metal chloride salt.Charge carriers may include ionic liquids or organic salts, for exampletetramethyl ammonium chloride, trimethylphenyl ammonium chloride,phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazoliumchloride. In the exemplary apparatus discussed above, the salt ispresent in the aqueous solution in the chamber. Potassium chloride(KCl), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture ofpotassium ferrocyanide and potassium ferricyanide is typically used.KCl, NaCl and a mixture of potassium ferrocyanide and potassiumferricyanide are preferred. The charge carriers may be asymmetric acrossthe membrane. For instance, the type and/or concentration of the chargecarriers may be different on each side of the membrane.

The salt concentration may be at saturation. The salt concentration maybe 3 M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M,from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to1.4 M. The salt concentration is preferably from 150 mM to 1 M. Themethod is preferably carried out using a salt concentration of at least0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M orat least 3.0 M. High salt concentrations provide a high signal to noiseratio and allow for currents indicative of the presence of a nucleotideto be identified against the background of normal current fluctuations.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is phosphate buffer. Other suitablebuffers are HEPES and Tris-HCl buffer. The methods are typically carriedout at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pHused is preferably about 7.5.

The methods may be carried out at from 0° C. to 100° C., from 15° C. to95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80°C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports enzyme function, such as about 37° C.

Molecular Brake The movement of the concatenated polynucleotide throughthe pore is preferably controlled by a molecular brake. The molecularbrake is preferably bound to the first target polynucleotide before itis contacted with the transmembrane pore and the protein controls themovement of the entire concatenated polynucleotide through the pore. Themolecular brake may be attached to a Y adaptor present on the firsttarget polynucleotide as discussed below. The one or more subsequentpolynucleotides preferably do not have a molecular brake bound to thembefore they are attached to the first target polynucleotide.

Any molecular brake may be used including any of those disclosed inInternational Application No. PCT/GB2014/052737 (published as WO2015/110777).

The molecular brake is preferably a polynucleotide binding protein. Thepolynucleotide binding protein may be any protein that is capable ofbinding to the polynucleotide and controlling its movement through thepore. It is straightforward in the art to determine whether or not aprotein binds to a polynucleotide. The protein typically interacts withand modifies at least one property of the polynucleotide. The proteinmay modify the polynucleotide by cleaving it to form individualnucleotides or shorter chains of nucleotides, such as di- ortrinucleotides. The moiety may modify the polynucleotide by orienting itor moving it to a specific position, i.e. controlling its movement.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. A polynucleotide handling enzyme is apolypeptide that is capable of interacting with and modifying at leastone property of a polynucleotide. The enzyme may modify thepolynucleotide by cleaving it to form individual nucleotides or shorterchains of nucleotides, such as di- or trinucleotides. The enzyme maymodify the polynucleotide by orienting it or moving it to a specificposition. The polynucleotide handling enzyme does not need to displayenzymatic activity as long as it is capable of binding thepolynucleotide and controlling its movement through the pore. Forinstance, the enzyme may be modified to remove its enzymatic activity ormay be used under conditions which prevent it from acting as an enzyme.Such conditions are discussed in more detail below.

The polynucleotide handling enzyme is preferably derived from anucleolytic enzyme. The polynucleotide handling enzyme used in theconstruct of the enzyme is more preferably derived from a member of anyof the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15,3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. Theenzyme may be any of those disclosed in International Application No.PCT/GB10/000133 (published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases, translocasesand topoisomerases, such as gyrases. Suitable enzymes include, but arenot limited to, exonuclease I from E. coli (SEQ ID NO: 11), exonucleaseIII enzyme from E. coli (SEQ ID NO: 13), RecJ from T. thermophilus (SEQID NO: 15) and bacteriophage lambda exonuclease (SEQ ID NO: 17), TatDexonuclease and variants thereof. Three subunits comprising the sequenceshown in SEQ ID NO: 15 or a variant thereof interact to form a trimerexonuclease. The polymerase may be PyroPhage® 3173 DNA Polymerase (whichis commercially available from Lucigen® Corporation), SD Polymerase(commercially available from Bioron®) or variants thereof. The enzyme ispreferably Phi29 DNA polymerase (SEQ ID NO: 9) or a variant thereof. Thetopoisomerase is preferably a member of any of the Moiety Classification(EC) groups 5.99.1.2 and 5.99.1.3.

The enzyme is most preferably derived from a helicase. The helicase maybe or be derived from a He1308 helicase, a RecD helicase, such as TraIhelicase or a TrwC helicase, a XPD helicase or a Dda helicase. Thehelicase may be or be derived from He1308 Mbu (SEQ ID NO: 18), He1308Csy (SEQ ID NO: 19), He1308 Tga (SEQ ID NO: 20), He1308 Mhu (SEQ ID NO:21), TraI Eco (SEQ ID NO: 22), XPD Mbu (SEQ ID NO: 23) or a variantthereof.

The helicase may be any of the helicases, modified helicases or helicaseconstructs disclosed in International Application Nos. PCT/GB2012/052579(published as WO 2013/057495); PCT/GB2012/053274 (published as WO2013/098562); PCT/GB2012/053273 (published as WO2013098561);PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924(published as WO 2014/013259); PCT/GB2013/051928 (published as WO2014/013262) and PCT/GB2014/052736 (published as WO/2015/055981).

The helicase preferably comprises the sequence shown in SEQ ID NO: 25(Trwc Cba) or as variant thereof, the sequence shown in SEQ ID NO: 18(He1308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 24(Dda) or a variant thereof. Variants may differ from the nativesequences in any of the ways discussed below for transmembrane pores. Apreferred variant of SEQ ID NO: 24 comprises (a) E94C and A360C or (b)E94C, A360C, C109A and C136A and then optionally (ΔM1)G1 (i.e. deletionof M1 and then addition G1). It may also be termed M1G. Any of thevariants discussed above may further comprise M1G.

The Dda helicase preferably comprises any of the modifications disclosedin International Application Nos. PCT/GB2014/052736 andPCT/GB2015/052916 (published as WO/2015/055981 and WO 2016/055777).

Any number of helicases may be used in accordance with the invention.For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may beused. In some embodiments, different numbers of helicases may be used.

The method of the invention preferably comprises contacting thepolynucleotide with two or more helicases. The two or more helicases aretypically the same helicase. The two or more helicases may be differenthelicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. Preferred helicase constructs for use in the invention aredescribed in International Application Nos. PCT/GB2013/051925 (publishedas WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259);PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

A variant of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or25 is an enzyme that has an amino acid sequence which varies from thatof SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 andwhich retains polynucleotide binding ability. This can be measured usingany method known in the art. For instance, the variant can be contactedwith a polynucleotide and its ability to bind to and move along thepolynucleotide can be measured. The variant may include modificationsthat facilitate binding of the polynucleotide and/or facilitate itsactivity at high salt concentrations and/or room temperature. Variantsmay be modified such that they bind polynucleotides (i.e. retainpolynucleotide binding ability) but do not function as a helicase (i.e.do not move along polynucleotides when provided with all the necessarycomponents to facilitate movement, e.g. ATP and Mg²⁺). Suchmodifications are known in the art. For instance, modification of theMg²⁺ binding domain in helicases typically results in variants which donot function as helicases. These types of variants may act as molecularbrakes (see below).

Over the entire length of the amino acid sequence of SEQ ID NO: 9, 11,13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25, a variant will preferablybe at least 50% homologous to that sequence based on amino acidsimilarity or identity. More preferably, the variant polypeptide may beat least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% and more preferably at least 95%,97% or 99% homologous based on amino acid similarity or identity to theamino acid sequence of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22,23, 24 or 25 over the entire sequence. There may be at least 80%, forexample at least 85%, 90% or 95%, amino acid similarity or identity overa stretch of 200 or more, for example 230, 250, 270, 280, 300, 400, 500,600, 700, 800, 900 or 1000 or more, contiguous amino acids (“hardhomology”). Homology is determined as described above. The variant maydiffer from the wild-type sequence in any of the ways discussed abovewith reference to SEQ ID NO: 2 and 4 above. The enzyme may be covalentlyattached to the pore. Any method may be used to covalently attach theenzyme to the pore.

A preferred molecular brake is TrwC Cba-Q594A (SEQ ID NO: 25 with themutation Q594A). This variant does not function as a helicase (i.e.binds polynucleotides but does not move along them when provided withall the necessary components to facilitate movement, e.g. ATP and Mg²⁺).

In strand sequencing, the polynucleotide is translocated through thepore either with or against an applied potential. Exonucleases that actprogressively or processively on double stranded polynucleotides can beused on the cis side of the pore to feed the remaining single strandthrough under an applied potential or the trans side under a reversepotential. Likewise, a helicase that unwinds the double stranded DNA canalso be used in a similar manner. A polymerase may also be used. Thereare also possibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand back through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

Any helicase may be used in the method. Helicases may work in two modeswith respect to the pore. First, the method is preferably carried outusing a helicase such that it moves the polynucleotide through the porewith the field resulting from the applied voltage. In this mode the 5′end of the polynucleotide is first captured in the pore, and thehelicase moves the polynucleotide into the pore such that it is passedthrough the pore with the field until it finally translocates through tothe trans side of the membrane. Alternatively, the method is preferablycarried out such that a helicase moves the polynucleotide through thepore against the field resulting from the applied voltage. In this modethe 3′ end of the polynucleotide is first captured in the pore, and thehelicase moves the polynucleotide through the pore such that it ispulled out of the pore against the applied field until finally ejectedback to the cis side of the membrane.

The method may also be carried out in the opposite direction. The 3′ endof the polynucleotide may be first captured in the pore and the helicasemay move the polynucleotide into the pore such that it is passed throughthe pore with the field until it finally translocates through to thetrans side of the membrane.

When the helicase is not provided with the necessary components tofacilitate movement or is modified to hinder or prevent its movement, itcan bind to the polynucleotide and act as a brake slowing the movementof the polynucleotide when it is pulled into the pore by the appliedfield. In the inactive mode, it does not matter whether thepolynucleotide is captured either 3′ or 5′ down, it is the applied fieldwhich pulls the polynucleotide into the pore towards the trans side withthe enzyme acting as a brake. When in the inactive mode, the movementcontrol of the polynucleotide by the helicase can be described in anumber of ways including ratcheting, sliding and braking. Helicasevariants which lack helicase activity can also be used in this way.

The polynucleotide may be contacted with the polynucleotide bindingprotein and the pore in any order. It is preferred that, when thepolynucleotide is contacted with the polynucleotide binding protein,such as a helicase, and the pore, the polynucleotide firstly forms acomplex with the protein. When the voltage is applied across the pore,the polynucleotide/protein complex then forms a complex with the poreand controls the movement of the polynucleotide through the pore.

Any steps in the method using a polynucleotide binding protein aretypically carried out in the presence of free nucleotides or freenucleotide analogues and an enzyme cofactor that facilitates the actionof the polynucleotide binding protein. The free nucleotides may be oneor more of any of the individual nucleotides discussed above. The freenucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP).The free nucleotides are preferably selected from AMP, TMP, GMP, CMP,UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferablyadenosine triphosphate (ATP). The enzyme cofactor is a factor thatallows the construct to function. The enzyme cofactor is preferably adivalent metal cation. The divalent metal cation is preferably Mg²⁺,Mn²⁺, Ca²⁺ or Co²⁺. The enzyme cofactor is most preferably Mg²⁺.

The molecular brakes may be any compound or molecule which binds to thepolynucleotide and slows the movement of the polynucleotide through thepore. The molecular brake may be any of those discussed above. Themolecular brake preferably comprises a compound which binds to thepolynucleotide. The compound is preferably a macrocycle. Suitablemacrocycles include, but are not limited to, cyclodextrins, calixarenes,cyclic peptides, crown ethers, cucurbiturils, pillararenes, derivativesthereof or a combination thereof. The cyclodextrin or derivative thereofmay be any of those disclosed in Eliseev, A. V., and Schneider, H-J.(1994) J Am. Chem. Soc. 116, 6081-6088. The cyclodextrin is morepreferably heptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD).

Spacers in the Target Polynucleotide

If a helicase is used in the invention, it may be stalled at one or morespacers as discussed in International Application No. PCT/GB2014/050175(published as WO 2014/135838). Any configuration of one or morehelicases and one or more spacers disclosed in the InternationalApplication may be used in this invention.

Double Stranded Polynucleotide

If a target polynucleotide is double stranded, the method preferablyfurther comprises providing the target polynucleotide with a hairpinloop at one end of the polynucleotide. The method may comprise linkingthe two strands of the target polynucleotide at one end with a hairpinloop. The pore and optionally the molecular brake preferably separatesthe two strands of the target polynucleotide and controls the movementof the target polynucleotide through the pore one strand at a time.Linking and interrogating both strands on a double stranded construct inthis way increases the efficiency and accuracy of characterisation.

Suitable hairpin loops can be designed using methods known in the art.The hairpin loop may be any length. The hairpin loop is typically 110 orfewer nucleotides, such as 100 or fewer nucleotides, 90 or fewernucleotides, 80 or fewer nucleotides, 70 or fewer nucleotides, 60 orfewer nucleotides, 50 or fewer nucleotides, 40 or fewer nucleotides, 30or fewer nucleotides, 20 or fewer nucleotides or 10 or fewernucleotides, in length. The hairpin loop is preferably from about 1 to110, from 2 to 100, from 5 to 80 or from 6 to 50 nucleotides in length.Longer lengths of the hairpin loop, such as from 50 to 110 nucleotides,are preferred if the loop is involved in the differential selectabilityof the adaptor. Similarly, shorter lengths of the hairpin loop, such asfrom 1 to 5 nucleotides, are preferred if the loop is not involved inthe selectable binding as discussed below.

The hairpin loop may be provided at either end of the polynucleotide,i.e. the 5′ or the 3′ end. The hairpin loop may be ligated to thepolynucleotide using any method known in the art. The hairpin loop maybe ligated using a ligase, such as T4 DNA ligase, E. coli DNA ligase,Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase.

The two strands of the polynucleotide may be separated using any methodknown in the art. For instance, they may be separated by a molecularbrake, such as a polynucleotide binding protein, or using conditionswhich favour dehybridsation (examples of conditions which favourdehybridisation include, but are not limited to, high temperature, highpH and the addition of agents that can disrupt hydrogen bonding or basepairing, such as formamide and urea).

The hairpin loop preferably comprises a selectable binding moiety. Thisallows the polynucleotide to be purified or isolated. A selectablebinding moiety is a moiety that can be selected on the basis of itsbinding properties. Hence, a selectable binding moiety is preferably amoiety that specifically binds to a surface. A selectable binding moietyspecifically binds to a surface if it binds to the surface to a muchgreater degree than any other moiety used in the invention. In preferredembodiments, the moiety binds to a surface to which no other moiety usedin the invention binds.

Suitable selective binding moieties are known in the art. Preferredselective binding moieties include, but are not limited to, biotin, apolynucleotide sequence, antibodies, antibody fragments, such as Fab andScSv, antigens, polynucleotide binding proteins, poly histidine tailsand GST tags. The most preferred selective binding moieties are biotinand a selectable polynucleotide sequence. Biotin specifically binds to asurface coated with avidins. Selectable polynucleotide sequencesspecifically bind (i.e. hybridise) to a surface coated with homologussequences. Alternatively, selectable polynucleotide sequencesspecifically bind to a surface coated with polynucleotide bindingproteins.

The hairpin loop and/or the selectable binding moiety may comprise aregion that can be cut, nicked, cleaved or hydrolysed. Such a region canbe designed to allow the polynucleotide to be removed from the surfaceto which it is bound following purification or isolation. Suitableregions are known in the art. Suitable regions include, but are notlimited to, an RNA region, a region comprising desthiobiotin andstreptavidin, a disulphide bond and a photocleavable region.

Leader Sequence

The target polynucleotide may be provided with a leader sequence whichpreferentially threads into the pore or which is capable of selectivelyattaching to the preceding target polynucleotide. The leader sequencefacilitates the method of the invention. The leader sequence may bedesigned to preferentially thread into the transmembrane pore andthereby facilitate the movement of polynucleotide through the pore. Theleader sequence may be designed such that it selectively attaches to thepreceding target polynucleotide and facilitates the formation of theconcatenated polynucleotide. The leader sequence can also be used tolink the polynucleotide to the one or more anchors as discussed above.

The leader sequence typically comprises a polymer. The polymer ispreferably negatively charged. The polymer is preferably apolynucleotide, such as DNA or RNA, a modified polynucleotide (such asabasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. Theleader preferably comprises a polynucleotide and more preferablycomprises a single stranded polynucleotide. The leader sequence cancomprise any of the polynucleotides discussed above. The single strandedleader sequence most preferably comprises a single strand of DNA, suchas a poly dT section. The leader sequence preferably comprises the oneor more spacers.

The leader sequence can be any length, but is typically 10 to 150nucleotides in length, such as from 20 to 150 nucleotides in length. Thelength of the leader typically depends on the transmembrane pore used inthe method.

The leader sequence in a subsequent target polynucleotide typicallycomprises the part which hybridizes to the part in the preceding targetpolynucleotide. The leader sequence preferably comprises one part of theclick chemistry, such as a click reactive group.

Y Adaptors

A double stranded target polynucleotide may be provided with adaptors atone or both ends. A double stranded target polynucleotide may beprovided with a Y adaptor at both ends.

A double stranded target polynucleotide may be provided with a Y adaptorat one end and a hairpin loop at the other end. A method ofcharacterising a polynucleotide may comprise attaching a Y adaptor toone end of a double stranded target polynucleotide and attaching ahairpin loop at the other end. The Y adaptor and/or the hairpin adaptorare typically polynucleotide adaptors. They may be formed from any ofthe polynucleotides discussed above.

The Y adaptor typically comprises (a) a double stranded region and (b) asingle stranded region or a region that is not complementary at theother end. The Y adaptor may be described as having an overhang if itcomprises a single stranded region. The presence of a non-complementaryregion in the Y adaptor gives the adaptor its Y shape since the twostrands typically do not hybridise to each other unlike the doublestranded portion. In other words, the Y adaptor comprises twopolynucleotide strands, e.g. DNA strands. A portion extending to the 3′end of the first strand is complementary to a portion extending to the5′ end of the other strand. The complementary portions hybridise to eachother and form a duplex, or double stranded region of the Y adaptor. Theremaining portions of the first and second strands are not complementaryand do not hybridise to one another.

In one embodiment, the Y adaptor comprises one or more attachment sitesthat can be used to selectively attach a first (preceding) targetpolynucleotide to a second (subsequent) target polynucleotide asdescribed herein.

In one embodiment, a first attachment site is present in the doublestranded region of the Y adaptor. When the Y adaptor is ligated to theend of a target polynucleotide, the attachment site in the doublestranded region is “hidden” until the target polynucleotide interactswith a pore. This “hidden” attachment site corresponds to the part ofthe first (preceding) target polynucleotide that can be selectivelyattached to a part of the second (subsequent) target polynucleotide.

In one embodiment, a second attachment site is present in the singlestranded region of the Y adaptor. When the Y adaptor is ligated to theend of a second target polynucleotide, the attachment site in the singlestranded region is available to bind to a first attachment site in afirst target polynucleotide when the first target polynucleotideinteracts with a pore to reveal the first attachment site. This“exposed” attachment site corresponds to the part of the second(subsequent) target polynucleotide that can be selectively attached to apart of the first (preceding) target polynucleotide.

A Y adaptor may comprise a first attachment site and a second attachmentsite as described above. In one embodiment, the first and secondattachment sites present in a Y adaptor are not complementary to eachother. A population of different Y adaptors, wherein complementaryattachment sites are present in different Y adaptors, may then be usedto perform a method as described herein. In an example of thisembodiment, the first (hidden) attachment site and the second (exposed)attachment site in a first Y adaptor may both have the same sequence,which sequence is complementary to the sequence of a first (hidden)attachment site and a second (exposed) attachment site in a second Yadaptor. The first and second Y adaptors may then be used together,wherein a first (preceding) target polynucleotide comprising the first Yadaptor is concatenated to a second (subsequent) target polynucleotidethat comprises the second Y adaptor. In another embodiment, the firstand second attachment sites present in a Y adaptor are complementary toeach other. In this embodiment, a single type of Y adaptor may be used.

The invention provides a Y adaptor comprising a first polynucleotidestrand and a second polynucleotide strand, wherein: (i) a portionextending to the 3′ end of the first polynucleotide strand iscomplementary to a portion extending to the 5′ end of the secondpolynucleotide strand and the complementary portions form a duplex; (ii)a portion extending to the 5′ end of the first polynucleotide strand anda portion extending to the 3′ end of the second polynucleotide strand donot hybridise to one another; and (iii) the portion extending to the 5′end of the second polynucleotide strand comprises a sequence that iscapable, when the duplex is unwound, of hybridising to a sequencecomprised in the a portion extending to the 5′ end of the firstpolynucleotide strand.

The sequence in the portion extending to the 5′ end of the secondpolynucleotide strand and the sequence comprised in the a portionextending to the 5′ end of the first polynucleotide strand that arecapable of hybridising to each other preferably have a length of from 6to 50 base pairs, such as from 7 to 40, 8 to 30, 9 to 20 or 10 to 15base pairs.

The invention also provides a Y adaptor comprising a firstpolynucleotide strand and a second polynucleotide strand, wherein: (i) aportion extending to the 3′ end of the first polynucleotide strand iscomplementary to a portion extending to the 5′ end of the secondpolynucleotide strand and the complementary portions form a duplex; (ii)a portion extending to the 5′ end of the first polynucleotide strand anda portion extending to the 3′ end of the second polynucleotide strand donot hybridise to one another; and (iii) the portion extending to the 5′end of the second polynucleotide strand comprises a sequence that isidentical to a sequence comprised in the a portion extending to the 5′end of the first polynucleotide strand.

The sequence in the portion extending to the 5′ end of the secondpolynucleotide strand that is identical to the sequence comprised in thea portion extending to the 5′ end of the first polynucleotide strandpreferably has a length of from 6 to 50 base pairs, such as from 7 to40, 8 to 30, 9 to 20 or 10 to 15 base pairs.

The duplex region in the Y adaptor may have a length of from about 6 to200 base pairs, such as from 10 to 150, 20 to 175, 25 to 150, 50 to 125or 75 to 100 base pairs. The duplex region may comprise a blockersequence to prevent movement of a helicase along the duplex strand. Inone embodiment, the blocker comprises iSp18. Suitable blocker sequencesare described in the Examples.

In the Y adaptor, the portion extending to the 5′ end of the firstpolynucleotide strand is preferably from 10 to 100 base pairs in length,such as from 10 to 75, 20 to 65 or 25 to 50 base pairs in length. Theportion extending to the 5′ end of the first polynucleotide strandcomprises an exposed attachment site. The portion extending to the 5′end of the first polynucleotide strand preferably comprises a polymerleader sequence. The polymer is preferably negatively charged. Thepolymer is preferably a polynucleotide, such as DNA or RNA, a modifiedpolynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG)or a polypeptide. The leader sequence preferably comprises one or morespacers, such as iSpC3. Examples of suitable leader sequences aredescribed in the Examples.

The leader sequence can be any length, but is typically 10 to 150nucleotides in length, such as from 20 to 150, 25 to 100 or 30 to 50nucleotides in length. The length of the leader typically depends on thetransmembrane pore used in the method. In one embodiment the combinedlength of the leader sequence and the exposed attachment site, which maycomprise all or part of the leader sequence is from 25 to 40, such as 30base pairs.

In one embodiment, the 5′ end of the first polynucleotide strand of theadaptor comprises comprises a first part of the click chemistry, such asa click reactive group.

In one embodiment, the 3′ end of the second polynucleotide strand of theadaptor comprises a second part of the click chemistry, such as a clickreactive group. The first part of the click chemistry is a part thatreacts with the second part of the click chemistry. Typically, in thisembodiment, the portion extending to the 5′ end of the secondpolynucleotide strand comprises a sequence that is capable, when theduplex is unwound, of hybridising to a sequence comprised in the aportion extending to the 5′ end of the first polynucleotide strand.

In another embodiment, the 3′ end of the second polynucleotide strand ofthe adaptor comprises a first part of the click chemistry, such as aclick reactive group. In this embodiment the adaptor is designed for usetogether with a second Y adaptor which comprises a second part of theclick chemistry, such as a click reactive group at the 5′ end of itsfirst polynucleotide strand and a second part of the click chemistry,such as a click reactive group at the 3′ end of its secondpolynucleotide strand. Typically, in this embodiment, the portionextending to the 5′ end of the second polynucleotide strand comprises asequence that is identical to a sequence comprised in the a portionextending to the 5′ end of the first polynucleotide strand.

In one embodiment, the 5′ end of the first polynucleotide strand of theadaptor comprises a phosphatase. The phosphatase may facilitate ligationof the 5′ end of the first polynucleotide strand to the 3′ end ofanother polynucleotide strand (typically the 3′ end of the second strandof a second adaptor after exposure of the hidden attachment site in thesecond adaptor).

The Y adaptor may comprise one or more anchors. Anchors are discussed inmore detail above. In one embodiment, the Y adaptor preferably comprisesan anchor attached to a third polynucleotide strand which comprises a 5′region that hybridises to the first polynucleotide strand. The part ofthe first polynucleotide strand to which the third polynucleotide strandbinds is typically between the region of the first polynucleotide strandthat hybridises to the second polynucleotide strand and the leadersequence. The anchor is preferably attached at or close to the 3′ end ofthe third polynucleotide strand. A fourth polynucleotide strand may behybridized to a region of the third polynucleotide strand that is nothybridised to the first polynucleotide strand. The anchor is preferablycholesterol.

One part of the non-complementary region in the second strand of each Yadaptor preferably forms a loop structure. This facilitates the methodof invention as discussed above. In particular it facilitates the actionof ligases that join double stranded polynucleotides. In thisembodiment, the two sides of the loop typically hybridise to oneanother. The nucleotide base at the 3′ end of the second polynucleotidestrand is preferably at the end of the loop. The nucleotide base at the3′ end of the second polynucleotide strand is typically hybridised toanother part of the second polynucleotide strand. The first (hidden)attachment site is preferably adjacent to, but not part of the loopstructure. Hybridisation of the first (hidden) attachment site to thesecond (exposed) attachment site in a different Y adaptor elongates theloop. The Y adaptors, which are at the ends of target polynucleotides,can then be joined, for example by ligation (using a ligase) or by clickchemistry. This embodiment results in the two adaptors (and hence thepreceding and subsequent target polynucleotides) being attached by aloop which may dehybridise and move through the pore.

The loop in the second polynucleotide strand may be any length. The loopin the second polynucleotide strand preferably comprises 10 to 50nucleotides, such as from 15 to 40, or 20 to 25, for example 23nucleotides.

The Y adaptor preferably comprises a leader sequence whichpreferentially threads into the pore. Leader sequences are discussedabove. In one embodiment a second (exposed) attachment site is presentin the same strand of the Y adaptor as a leader sequence. Typically, theleader sequence is at the 5′ end of the Y adaptor and the secondattachment site is present between the leader sequence and the doublestranded region. The exposed attachment site may form part of the leadersequence.

The Y adaptor may further comprise one or more polynucleotide bindingproteins. In one embodiment a polynucleotide binding protein is attachedto the first polynucleotide strand of the Y adaptor. The polynucleotidebinding protein is preferably located 3′ of the leader sequence and 5′to the blocker sequence. The polynucleotide binding protein ispreferably a helicase. More than one, such as two or more polynucleotidebinding proteins, preferably two or more helicases, may be comprised inthe adaptor. The two or more polynucleotide binding proteins arepreferably different from one another, for example are two differenthelicases. Where two polynucleotide binding proteins are present, thetwo proteins preferably process the bound polynucleotide at differentrates once the effect of the blocker sequence is removed. For example, afirst polynucleotide binding protein, preferably a first helicase, maybe attached to the first polynucleotide strand of the adaptor 3′ to asecond polynucleotide binding protein, preferably a second helicase. Inthis example, the first polynucleotide binding protein may separate thetwo strands of a double stranded target polynucleotide and the secondpolynucleotide binding protein may act to control movement of the targetpolynucleotide through a nanopore. In this embodiment, the firstpolynucleotide binding protein may be referred to as a “release protein”and the second polynucleotide binding protein as a “motor protein”. Therelease protein preferably moves along the polynucleotide faster thanthe motor protein.

The Y adaptor preferably comprises a selectable binding moiety asdiscussed above. The Y adaptor and/or the selectable binding moiety maycomprise a region that can be cut, nicked, cleaved or hydrolysed asdiscussed above.

The Y adaptor and/or the hairpin loop may be ligated to thepolynucleotide using any method known in the art. One or both of theadaptors may be ligated using a ligase, such as T4 DNA ligase, E. coliDNA ligase, Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase.Alternatively, the adaptors may be added to the polynucleotide using themethods of the invention discussed below.

In a preferred embodiment, the method comprises modifying the doublestranded polynucleotide so that it comprises adaptors at its ends. Forexample, in one embodiment, the method comprises modifying the doublestranded polynucleotide so that it comprises the Y adaptor at one endand the hairpin loop at the other end. In another embodiment, the methodcomprises modifying the double stranded polynucleotide so that itcomprises a Y adaptor at each end. Any manner of modification can beused. The method preferably comprises modifying the double strandedpolynucleotide in accordance with the invention. This is discussed inmore detail below. The methods of modification and characterisation maybe combined in any way.

Adding Hairpin Loops and Leader Sequences

The double stranded polynucleotide may be provided with Y adaptors andhairpin loops by contacting the polynucleotide with a MuA transposaseand a population of double stranded MuA substrates, wherein a proportionof the substrates in the population are Y adaptors comprising the leadersequence and wherein a proportion of the substrates in the populationare hairpin loops. The transposase fragments the double strandedpolynucleotide and ligates MuA substrates to one or both ends of thefragments. This produces a plurality of modified double strandedpolynucleotides comprising the leader sequence at one end and thehairpin loop at the other. The modified double stranded polynucleotidesmay then be investigated using the method of the invention.

These MuA based methods are disclosed in International Application No.PCT/GB2014/052505 published as (WO 2015/022544). They are also discussedin detail in International Application No PCT/GB2015/050991.

The double stranded polynucleotide may be provided with Y adaptors bycontacting the polynucleotide with a MuA transposase and a population ofdouble stranded MuA substrates, wherein the substrates are Y adaptorscomprising a leader sequence and attachment sites. The transposasefragments the double stranded polynucleotide and ligates MuA substratesto one or both ends of the fragments. This produces a plurality ofmodified double stranded target polynucleotides comprising singlestranded overhangs at both ends. The modified double strandedpolynucleotides may then be investigated using the method of theinvention.

Modified Polynucleotide Analytes

Before characterisation, the polynucleotide may be modified bycontacting the polynucleotide with a polymerase and a population of freenucleotides under conditions in which the polymerase forms a modifiedpolynucleotide using the polynucleotide as a template, wherein thepolymerase replaces one or more of the nucleotide species in thepolynucleotide with a different nucleotide species when forming themodified polynucleotide analyte. The modified polynucleotide may then becharacterised in accordance with the invention. This type ofmodification is described in PCT Application No. PCT/GB2015/050483. Anyof the polymerases discussed above may be used. The polymerase ispreferably Klenow or 90 North.

Population of Y Adaptors

The invention also provides a population of two or more polynucleotide Yadaptors, wherein each adaptor comprises first and second parts (firstand second attachment sites) which are capable of hybridising togetherand wherein each first part (first attachment site) is initiallyprotected from hybridisaton to the second part (second attachment site).The population may comprise any number of adaptors, such as the numbersdiscussed above for target polynucleotide.

Also provided is a population of polynucleotide adaptors comprising afirst polynucleotide adaptor and a second polynucleotide adaptor,wherein the first polynucleotide adaptor and the second polynucleotideadaptor each comprise a first polynucleotide strand and a secondpolynucleotide strand, wherein: (a)(i) a portion extending to the 3′ endof the first polynucleotide strand is complementary to a portionextending to the 5′ end of the second polynucleotide strand and thecomplementary portions form a duplex; (ii) a portion extending to the 5′end of the first polynucleotide strand and a portion extending to the 3′end of the second polynucleotide strand do not hybridise to one another;and wherein (b) the portion extending to the 5′ end of the secondpolynucleotide strand of the first polynucleotide adaptor comprises asequence that is capable, when the duplex is unwound, of hybridising toa sequence comprised in the portion extending to the 5′ end of the firstpolynucleotide strand of the second polynucleotide adaptor. Thesepolynucleotide adaptors are referred to a “Y adaptors” because of theirshape.

The Y adaptors in the population may have any of the features describedin the section “Y adaptors” above. Any of the embodiments discussedabove with reference to the methods of the invention equally apply tothe population of the invention.

As discussed above, each Y adaptor typically comprises (a) a doublestranded region and (b) a single stranded region or a region that is notcomplementary at the other end. The presence of the non-complementaryregion in the Y adaptor gives the adaptor its Y shape since the twopart/strands typically do not hybridise to each other unlike the doublestranded portion.

In other words, the Y adaptor comprises two polynucleotide strands, e.g.DNA strands. A portion extending to the 3′ end of the first strand iscomplementary to a portion extending to the 5′ end of the other strand.The complementary portions hybridise to each other and form a duplex, ordouble stranded region of the Y adaptor. The remaining portions of thefirst and second strands are not complementary and do not hybridise toone another.

In one embodiment, the Y adaptor comprises one or more attachment sitesthat can be used to selectively attach a first (preceding) targetpolynucleotide to a second (subsequent) target polynucleotide asdescribed herein.

In one embodiment, a first attachment site is present in the doublestranded region of the Y adaptor. When the Y adaptor is ligated to theend of a target polynucleotide, the attachment site in the doublestranded region is “hidden” until the target polynucleotide interactswith a pore. This “hidden” attachment site corresponds to the part ofthe first (preceding) target polynucleotide that can be selectivelyattached to a part of the second (subsequent) target polynucleotide.

In one embodiment, a second attachment site is present in the singlestranded region of the Y adaptor. When the Y adaptor is ligated to theend of a second target polynucleotide, the attachment site in the singlestranded region is available to bind to a first attachment site in afirst target polynucleotide when the first target polynucleotideinteracts with a pore to reveal the first attachment site. This“exposed” attachment site corresponds to the part of the second(subsequent) target polynucleotide that can be selectively attached to apart of the first (preceding) target polynucleotide.

A Y adaptor may comprise a first attachment site and a second attachmentsite as described above. In one embodiment, the first and secondattachment sites present in a Y adaptor are not complementary to eachother. A population of different Y adaptors, wherein complementaryattachment sites are present in different Y adaptors, may then be usedto perform a method as described herein. In another embodiment, thefirst and second attachment sites present in a Y adaptor arecomplementary to each other. In this embodiment, a single type of Yadaptor may be used.

The Y adaptor may comprise one or more anchors. Anchors are discussed inmore detail above. In one embodiment, the Y adaptor preferably comprisesan anchor attached to the portion extending to the 3′ end of the secondpolynucleotide strand.

The first part (first attachment site) is preferably initially protectedby hybridisaton to the opposite strand in the double stranded region ofthe Y adaptor.

One half of the non-complementary region in each Y adaptor preferablyforms a loop structure. This facilitates the method of invention asdiscussed above. The first part (is preferably adjacent to, but not partof the loop structure. Hybridisation of the first part to the secondpart in a different Y adaptor elongates the loop. This embodimentresults in the two adaptors (and hence the preceding and subsequenttarget polynucleotides) being attached by a loop which may dehybridiseand move through the pore.

Each Y adaptor preferably further comprises a leader sequence comprisingthe second part. Suitable leader sequences are discussed above. Thesecond part is preferably an overhang formed by a bridgingpolynucleotide hybridised to the free end of the leader sequence. Thebridging polynucleotide may be any length and formed from any of thetypes of polynucleotide discussed above. In one embodiment the second(exposed) attachment site is present in the same strand of the Y adaptoras a leader sequence. Typically, the leader sequence is at the 5′ end ofthe Y adaptor and the second attachment site is present between theleader sequence and the double stranded region.

In a preferred embodiment, only one of the adaptors in the populationcomprises a molecular brake. Attachment of this adaptor to the firsttarget polynucleotide means that the molecular brake will control themovement of the concatenated polynucleotide, i.e. all of the targetpolynucleotides, through the pore.

The first part preferably comprises a click reactive group and thesecond part comprises the complementary click reactive group. Thereactive groups may be any of those discussed above.

Kits

The present invention also provides a kit for characterising two or moredouble stranded target polynucleotides. In one embodiment, the kitcomprises a population of Y adaptors of the invention. In a furtherembodiment, the kit comprises a Y adaptor of the invention. In anotherembodiment, the kit comprises a population of Y adaptors of theinvention and a population of hairpin loops. Such loops are discussedabove.

The kit may further comprise a microparticle for delivering the targetpolynucleotides to a transmembrane pore in a membrane. The kit mayfurther comprise one or more anchors which are capable of coupling thepolynucleotides to a membrane. The microparticle and the one or moreanchors may be any of those discussed above with reference to the methodof the invention. The microparticle is preferably part of the kit forextracting and/or purifying the polynucleotide.

Any of the embodiments discussed above with reference to the method ofthe invention equally apply to the kits. The kit may further comprisethe components of a membrane, such as the components of an amphiphiliclayer or a triblock copolymer membrane. The kit may further comprise atransmembrane protein pore.

The kit of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. The kit may comprise a magnet or anelectromagnet. Such reagents or instruments include one or more of thefollowing: suitable buffer(s) (aqueous solutions), means to obtain asample from a subject (such as a vessel or an instrument comprising aneedle), means to amplify and/or express polynucleotides, a membrane asdefined above or voltage or patch clamp apparatus. Reagents may bepresent in the kit in a dry state such that a fluid sample resuspendsthe reagents. The kit may also, optionally, comprise instructions toenable the kit to be used in the method of the invention or detailsregarding for which organism the method may be used.

The following Examples illustrate the invention.

EXAMPLES Example 1

This example describes a method of characterising a concatenatedpolynucleotide where the method of attachment used to join thepolynucleotides together is by ligation.

Materials and Methods Ligation Prep

A leader strand (ID 1 below), a bottom strand (ID NO: 2 below),containing a 3′ hairpin, and a blocker strand (ID No: 3 below) wereannealed at 5 μM, 6 μM and 6 μM respectively in 50 mM HEPES pH 8, 100 mMpotassium acetate from 95° C. to 22° C. at 2° C. per minute. Thehybridised DNA was known as adapter 1.

An aliquot of T4 Dda-(E94C/F98W/C109A/C136A/A360C) (SEQ ID NO: 24 withmutations E94C/F98W/C109A/C136A/A360C and then (ΔM1)G1G2 (where(ΔM1)G1G2=deletion of M1 and then addition G1 and G2) was thawed on icebefore 50 ul was buffer exchanged into 50 mM HEPES pH 8, 100 mMpotassium acetate, 2 mM EDTA through a 0.5 ml Zeba column, according tothe manufacturer's instructions. The recovered protein was quantifiedusing the A280 nm value and adjusted to 0.25 mg ml⁻¹ using the samebuffer. 27 ul of buffer exchanged protein was mixed with 3 ul of adapter1 in a DNA low bind eppendorf and left to incubate for 10 mins at 35° C.0.37 ul of 8.1 mM TMAD was then added and the sample was left toincubate for 60 mins at 35° C. 30 ul of 50 mM HEPES pH 8, 1 M NaCl, 2 mMMgCl2, 2 mM rATP was then added and left for a further 20 mins at roomtemperature.

222 ul of Agencourt AMPure beads (Beckman Coulter) were then added andthe sample incubated for 5 mins at room temperature on a rotator. Thebeads were pelleted on a magnetic rack and the supernatant removed.While still on the magnetic rack the beads were washed with 500 ul of 50mM Tris pH 7.5, 2.5 M NaCl, 20% PEG 8,000, turning through 360° to bathethe pellet on the rack. The wash buffer was removed and the pelletpulsed briefly in a centrifuge before returning to the magnetic rack toremove the last remnants of solution. The pellet was then resuspended in30 ul of 50 mM Tris pH 7.5, 20 mM NaCl for 5 mins at room temperaturebefore being placed on a magnetic rack to recover the purified adapterwhich was known as preloaded Y-adapter 1.

500 ng of end-repaired and dA-tailed E. coli genomic DNA was ligated for10 mins at room temperature in 50 ul with 5 ul of 200 nM preloadedY-adapter 1 from above and 1 ul of 1 μM HP-adapter (ID NO: 4), in 1×Blunt/TA master mix (NEB). After incubation 0.5 ul of 5 μM hairpintether (ID NO: 6) was added and the sample left for a further 10 mins atroom temperature.

25 ul of MyOne C1 Streptavidin beads (Invitrogen) were bound to amagnetic rack and the supernatant was removed. The pellet was thenwashed 3× by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1mM EDTA. Finally the beads were resuspended in 50 ul of 10 mM Tris pH7.5, 2 M NaCl, 0.1 mM EDTA. The beads were then added to the ligationmix and incubated on a rotator for 10 mins at room temperature. The beadbound library was then added to a magnetic rack and the supernatant wasremoved. The pellet was then washed 3× by resuspension in 200 ul of 10mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads wereresuspended in 12.5 ul of 40 mM CAPS pH 10, 40 mM KCl, 5 mM Biotin, 0.1mM EDTA and 400 nM of tether (ID NO: 5). The sample was incubated for 10mins at 37° C. before the beads were pelleted and the library containingthe supernatant was removed.

Electrical measurements were acquired from single MspA nanoporesinserted in block co-polymer in buffer (25 mM K Phosphate buffer, 150 mMPotassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH8.0). After achieving a single pore inserted in the block co-polymer,then buffer (2 mL, 25 mM K Phosphate buffer, 150 mM PotassiumFerrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0) wasflowed through the system to remove any excess MspA nanopores. 500 ul of25 mM potassium phosphate buffer pH 8, 500 mM KCl, 2 mM MgCl2 and 2 mMrATP, with 10 mins between each wash. 12 ul of the recovered beadpurified library was added to 150 ul of 50 mM potassium phosphate bufferpH 8, 1 M KCl, 8 ul of 75 mM MgCl2, 75 mM rATP, 124 ul of nuclease freewater and 6 ul of T3 DNA ligase (NEB). 150 ul of this sequencing mix wasthen added to the nanopore system. The experiment was run at −140 mV andhelicase-controlled DNA movement monitored.

ID NO: 1 (SEQ ID NO: 28) /5Phos/GCGGTTGTT/iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3//iSpC3/(SEQ ID NO: 29) /iSp18//iSp18//iSp18//iSp18/ ID NO: 2 (SEQ ID NO: 30)/5Phos/ (SEQ ID NO: 31) /iSp18//iSp18//iSp18/ (SEQ ID NO: 32)/iSp18//iSp18//iSp18/ ID NO: 3 (SEQ ID NO: 33)/5BNA-GMBNA-GMBNA-T/ABNA-T//iBNA-A/ ID NO: 4 (SEQ ID NO: 34) /5Phos/(SEQ ID NO: 35) /iSp18//iSp18//iSp18/ (SEQ ID NO: 36)/iSp18//iSp18//iSp18/ ID NO: 5 (SEQ ID NO: 37) /5CholTEG/  ID NO: 6(SEQ ID NO: 38) /5desthiobiotinTEG/TT/iSp18//iSp18//iSp18//iSp18//iSp18//iSp18/ /iSp18//iSp18//iSp18//iSp18//iSp18//iSp18/TT/ 3CholTEG

Results

The helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) was used to controlthe movement of the concatenated polynucleotide through the MspAnanopore. FIGS. 2-6 each show a current trace (top trace on each slide)of a concatenated polynucleotide (a first polynucleotide attached to onesubsequent polynucleotide) as it translocated through the nanopore. Thelower trace of FIGS. 2-6 show zoomed in regions 1-5 of the upper trace.The lower traces show translocation of spacer groups (found in theleader and hairpin regions of the first and subsequent polynucleotideand marked with a *) through the nanopore. The spacer groups allowedmore current to flow through the nanopore as they translocated throughit. The example trace shows that the first polynucleotide wassuccessfully ligated to the subsequent polynucleotide.

Example 2

This example describes a method of characterising a concatenatedpolynucleotide where the method of attachment used to join thepolynucleotides together is by click chemistry (see FIG. 7 for a cartoonrepresentation of attachment using click chemistry).

Materials and Methods Click Ligation Prep:

A leader strand (ID NO: 7) and a blocker strand (ID NO: 8), containing atether hybridisation site, were annealed at 5.5 μM and 6 μM respectivelyin 50 mM HEPES pH 8, 100 mM potassium acetate from 95° C. to 22° C. at2° C. per minute. 10 μM of a bottom strand (ID NO: 9), containing a 3′hairpin, was heated to 95° C. for 1 min before being snap cooled on icein 50 mM HEPES pH 8, 100 mM potassium acetate. The two samples wereequilibrated to 50° C. before being mixed 1:1 and left at 40° C. for 1min before snap cooling on ice. The hybridised DNA was known as adapter2.

An aliquot of T4 Dda-(E94C/F98W/C109A/C136A/A360C) (SEQ ID NO: 24 withmutations E94C/F98W/C109A/C136A/A360C and then (ΔM1)G1G2 (where(ΔM1)G1G2=deletion of M1 and then addition G1 and G2) was thawed on icebefore 50 ul was buffer exchanged into 50 mM HEPES pH 8, 100 mMpotassium acetate, 2 mM EDTA through a 0.5 ml Zeba column, according tothe manufacturer's instructions. The recovered protein was quantifiedusing the A280 nm value and adjusted to 0.25 mg ml⁻¹ using the samebuffer.

27 ul of buffer exchanged protein was mixed with 3 ul of adapter 2 in aDNA low bind eppendorf and left to incubate for 10 mins at 35° C. 0.37ul of 8.1 mM TMAD was then added and the sample was left to incubate for60 mins at 35° C. 30 ul of 50 mM HEPES pH 8, 1 M NaCl, 2 mM MgCl2, 2 mMrATP was then added and left for a further 20 mins at room temperature.

222 ul of Agencourt AMPure beads (Beckman Coulter) were added and thesample incubated for 5 mins at room temperature on a rotator. The beadswere pelleted on a magnetic rack and the supernatant removed. Whilestill on the magnetic rack the beads were washed with 500 ul of 50 mMTris pH 7.5, 2.5 M NaCl, 20% PEG 8,000, turning through 360° to bathethe pellet on the rack. The wash buffer was removed and the pelletpulsed briefly in a centrifuge before returning to the magnetic rack toremove the last remnants of solution. The pellet was resuspended in 30ul of 50 mM Tris pH 7.5, 20 mM NaCl for 5 mins at room temperaturebefore being placed on a magnetic rack to recover the purified adapterwhich was known as preloaded Y-adapter 2.

500 ng of end-repaired and dA-tailed E. coli genomic DNA was ligated for10 mins at room temperature in 50 ul with 5 ul of 200 nM preloadedY-adapter 2 from above and 1 ul of 1 uM HP-adapter (ID NO: 4), in 1×Blunt/TA master mix (NEB). After incubation 0.5 ul of 5 μM hairpintether (ID NO: 6) was added and the sample left for a further 10 mins atroom temperature.

25 ul of MyOne C1 Streptavidin beads (Invitrogen) were bound to amagnetic rack and supernatant was removed. The pellet was then washed 3×by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA.Finally the beads were resuspended in 50 ul of 10 mM Tris pH 7.5, 2 MNaCl, 0.1 mM EDTA. The beads were then added to the ligation mix andincubated on a rotator for 10 mins at room temperature. The bead boundlibrary was then added to a magnetic rack and supernatant was removed.The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 12.5ul of 40 mM CAPS pH 10, 40 mM KCl, 5 mM Biotin, 0.1 mM EDTA and 400 nMof tether (ID NO: 5). The sample was incubated for 10 mins at 37° C.before the beads pelleted and the library containing supernatant wasremoved.

Electrical measurements were acquired from single MspA nanoporesinserted in block co-polymer in buffer (25 mM K Phosphate buffer, 150 mMPotassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH8.0). After achieving a single pore inserted in the block co-polymer,then buffer (2 mL, 25 mM K Phosphate buffer, 150 mM PotassiumFerrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0) wasflowed through the system to remove any excess MspA nanopores. 500 ul of25 mM potassium phosphate buffer pH 8, 500 mM KCl, 2 mM MgCl2 and 2 mMrATP, with 10 mins between each wash. 12 ul of the recovered beadpurified library was added to 150 ul of 50 mM potassium phosphate bufferpH 8, 1 M KCl, 8 ul of 75 mM MgCl2, 75 mM rATP and 130 ul of nucleasefree water. 150 ul of this sequencing mix was then added to the nanoporesystem. The experiment was run at −140 mV and helicase-controlled DNAmovement monitored.

ID NO: 7 (SEQ ID NO: 39) /Azide/ (SEQ ID NO: 28)/iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3/  (SEQ ID NO: 29)/iSp18//iSp18//iSp18//iSp18/  ID NO: 8 SEQ ID NO: 40 ID NO: 9(SEQ ID NO: 41) /5Phos/ (SEQ ID NO: 32) /iSp18//iSp18//iSp18//iSp18//DBCO/

Results

The helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) was used to controlthe movement of the concatenated polynucleotide through the MspAnanopore. FIG. 8 shows a current trace (top trace) of a concatenatedpolynucleotide (a first polynucleotide attached to five subsequentpolynucleotides) as it translocated through the nanopore. The lowertraces of FIG. 8 show zoomed in regions a*-k* of the upper trace. Thelower traces show translocation of spacer groups (found in the leaderand hairpin regions of the first and subsequent polynucleotides andmarked with an a*-k*) through the nanopore. The spacer groups allowedmore current to flow through the nanopore as they translocated throughit. The example trace shows that the first polynucleotide wassuccessfully attached to five subsequent polynucleotides using clickchemistry.

Example 3

This example describes a method of characterising a concatenatedpolynucleotide where the method of attachment used to join thepolynucleotides together is by click chemistry. In this example onegroup of adapters has a pre-bound enzyme which was used to produce aseed library and the second group has no enzyme bound which was used toproduce a sequencing library.

Seed Library Prep:

A seed library Y-adapter was produced using a leader strand (ID NO: 10),a bottom strand (ID NO: 9), containing a 3′ hairpin, and a blockerstrand (ID NO: 8), containing a tether hybridisation site, which wereannealed at 5.5 μM, 6 μM and 5 μM respectively in 50 mM HEPES pH 8, 100mM potassium acetate from 95° C. to 22° C. at 2° C. per minute. Thehybridised DNA was known as adapter 2.

An aliquot of T4 Dda-(E94C/F98W/C109A/C136A/A360C) (SEQ ID NO: 24 withmutations E94C/F98W/C109A/C136A/A360C and then (ΔM1)G1G2 (where(ΔM1)G1G2=deletion of M1 and then addition G1 and G2) was thawed on icebefore 50 ul was buffer exchanged into 50 mM HEPES pH 8, 100 mMpotassium acetate, 2 mM EDTA through a 0.5 ml Zeba column, according tothe manufacturer's instructions. The recovered protein was quantifiedusing the A280 nm value and adjusted to 0.25 mg ml⁻¹ using the samebuffer.

27 ul of buffer exchanged protein was mixed with 3 ul of adapter 2 in aDNA low bind eppendorf and left to incubate for 10 mins at 35° C. 0.37ul of 8.1 mM TMAD was then added and the sample was left to incubate for60 mins at 35° C. 30 ul of 50 mM HEPES pH 8, 1 M NaCl, 2 mM MgCl2, 2 mMrATP was then added and left for a further 20 mins at room temperature.

222 ul of Agencourt AMPure beads (Beckman Coulter) were added and thesample incubated for 5 mins at room temperature on a rotator. The beadswere pelleted on a magnetic rack and the supernatant removed. Whilestill on the magnetic rack the beads were washed with 500 ul of 50 mMTris pH 7.5, 2.5 M NaCl, 20% PEG 8,000, turning through 360° to bathethe pellet on the rack. The wash buffer was removed and the pelletpulsed briefly in a centrifuge before returning to the magnetic rack toremove the last remnants of solution. The pellet was resuspended in 30ul of 50 mM Tris pH 7.5, 20 mM NaCl for 5 mins at room temperaturebefore being placed on a magnetic rack to recover the purified adapterwhich was known as preloaded Seed Y-adapter 2.

A sequencing library Y-adapter was produced by hybridising a leaderstrand (ID NO: 7) and a blocker strand (ID NO: 11), containing a tetherhybridisation site and a polyA 5′ extension, at 250 nM and 300 nMrespectively in 50 mM HEPES pH 8, 100 mM potassium acetate from 95° C.to 22° C. at 2° C. per minute. 400 nM of a bottom strand (ID NO: 9),containing a 3′ hairpin, was heated to 95° C. for 1 min before beingsnap cooled on ice in 50 mM HEPES pH 8, 100 mM potassium acetate. Thetwo samples were equilibrated to 50° C. before being mixed 1:1 and leftat 40° C. for 1 min before snap cooling on ice. This sample was known asenzyme free sequencing library Y-adapter 3.

A seed library was set up by ligating 500 ng of end-repaired anddA-tailed E. coli genomic DNA for 10 mins at room temperature in 50 ulwith 5 ul of 200 nM preloaded seed Y-adapter 2 from above and 1 ul of 1μM HP-adapter (ID NO: 4), in 1× Blunt/TA master mix (NEB). Afterincubation 0.5 ul of 5 μM hairpin tether (ID NO: 6) was added and thesample left for a further 10 mins at room temperature.

25 ul of MyOne C1 Streptavidin beads (Invitrogen) were bound to amagnetic rack and supernatant was removed. The pellet was then washed 3×by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA.Finally the beads were resuspended in 50 ul of 10 mM Tris pH 7.5, 2 MNaCl, 0.1 mM EDTA. The beads were then added to the ligation mix andincubated on a rotator for 10 mins at room temperature. The bead boundlibrary was then added to a magnetic rack and supernatant was removed.The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 12.5ul of 40 mM CAPS pH 10, 40 mM KCl, 5 mM Biotin, 0.1 mM EDTA and 400 nMof tether (ID NO: 5). The sample was incubated for 10 mins at 37° C.before the beads were pelleted and the library containing supernatantwas removed.

A sequencing library was set up by ligating 500 ng of end-repaired anddA-tailed E. coli genomic DNA for 10 mins at room temperature in 50 ulwith 5 ul of 200 nM enzyme free sequencing library Y-adapter 3 fromabove and 1 ul of 1 μM HP-adapter (ID NO: 4), in 1× Blunt/TA master mix(NEB). After incubation 0.5 ul of 5 μM hairpin tether (ID NO: 6) wasadded and the sample left for a further 10 mins at room temperature.

25 ul of MyOne C1 Streptavidin beads (Invitrogen) were bound to amagnetic rack and supernatant was removed. The pellet was then washed 3×by resuspension in 200 ul of 10 mM Tris pH 7.5, 2 M NaCl, 0.1 mM EDTA.Finally the beads were resuspended in 50 ul of 10 mM Tris pH 7.5, 2 MNaCl, 0.1 mM EDTA. The beads were then added to the ligation mix andincubated on a rotator for 10 mins at room temperature. The bead boundlibrary was then added to a magnetic rack and supernatant was removed.The pellet was then washed 3× by resuspension in 200 ul of 10 mM Tris pH7.5, 2 M NaCl, 0.1 mM EDTA. Finally the beads were resuspended in 12.5ul of 40 mM CAPS pH 10, 40 mM KCl, 5 mM Biotin, 0.1 mM EDTA and 400 nMof tether (ID NO: 5). The sample was incubated for 10 mins at 37° C.before the beads were pelleted and the library containing supernatantwas removed.

Electrical measurements were acquired from single MspA nanoporesinserted in block co-polymer in buffer (25 mM K Phosphate buffer, 150 mMPotassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH8.0). After achieving a single pore inserted in the block co-polymer,then buffer (2 mL, 25 mM K Phosphate buffer, 150 mM PotassiumFerrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0) wasflowed through the system to remove any excess MspA nanopores. 500 ul of25 mM potassium phosphate buffer pH 8, 500 mM KCl, 2 mM MgCl2 and 2 mMrATP, with 10 mins between each wash. 6 ul of the recovered beadpurified seed library was added to 150 ul of 50 mM potassium phosphatebuffer pH 8, 1 M KCl, 8 ul of 75 mM MgCl2, 75 mM rATP and 130 ul ofnuclease free water. Following thorough mixing by inversion, 6 ul of thesequencing library was added and the sample again mixed thoroughly byinversion. 150 ul of this sequencing mix was then added to the nanoporesystem. The experiment was run at −140 mV and helicase-controlled DNAmovement monitored for 6 hours.

ID NO: 10 (SEQ ID NO: 28)/5SpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3// iSpC3//iSpC3/(SEQ ID NO: 29) /iSp18//iSp18//iSp18//iSp18/  ID NO 11 (SEQ ID NO: 42)

Results

The helicase T4 Dda-(E94C/F98W/C109A/C136A/A360C) was used to controlthe movement of the concatenated polynucleotide through the MspAnanopore. Similar results were obtained as were observed for example 2.

Example 4

This example describes a method of characterising and concatenating onlytemplate strands of target polynucleotides, where the method ofattachment used to join the polynucleotides together is by clickchemistry.

A concatenation adapter complex that contains a motor protein and arelease protein is prepared. This adapter is then ligated to both endsof a target polynucleotide. Both proteins are stalled on the ligatedadapter complex until the adaptor ligated to a target polynucleotide iscaptured by the pore. Once a first polynucleotide has been captured, theblocking chemistry used to stall the proteins is overcome by bothproteins. The motor protein then controls the interaction of the firstpolynucleotide with the pore, as described previously, and the releaseprotein, which can translocate more quickly than the motor protein,separates the strands to expose a sequence (3′ hybridisation site) inthe 3′ end of the adaptor linked to the end of the target polynucleotidethat is complementary to a 5′ nucleic acid sequence (5′ hybridisationsite) of the leader strand of an adapter complex that is ligated to asecond target polynucleotide. With the 3′ hybridisation site revealed,the 5′ hybridisation site in the second target polynucleotide can thenhybridise to the revealed 3′ hybridisation site and covalent coupling ofthe 3′ end of the first polynucleotide to the 5′ of a the secondpolynucleotide can occur (FIG. 10). This process then repeats forfurther concatenation of target polynucleotides.

1. A method of characterising two or more target polynucleotides,comprising: (a) contacting a first target polynucleotide with atransmembrane pore in a membrane such that the first targetpolynucleotide moves through the pore; (b) sequentially attaching to thefirst target polynucleotide one or more subsequent targetpolynucleotides to provide a concatenated polynucleotide within whichthe target polynucleotides move through the pore in attachment order,wherein a subsequent target polynucleotide is selectively attached tothe preceding target polynucleotide in the attachment order when thepreceding target polynucleotide moves through the pore; and (c) takingone or more measurements which are indicative of one or morecharacteristics of the concatenated polynucleotide as it moves withrespect to the pore.
 2. A method according to claim 1, wherein 2 ormore, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 ormore, 1,000 or more, 5,000 or more, 10,000 or more, 50,000 or more,100,000 or more, 500,000 or more, 1,000,000 or more or 5,000,000 or moresubsequent target polynucleotides are attached to the first targetpolynucleotide.
 3. A method according to claim 1, wherein a part of thepreceding target polynucleotide is initially protected from attachmentto the subsequent target polynucleotide and is revealed for attachmentas the preceding target polynucleotide moves through the pore.
 4. Amethod according to claim 1, wherein a part of the subsequent targetpolynucleotide selectively hybridises to a part of the precedingpolynucleotide and further wherein the part of the preceding targetpolynucleotide is initially protected from hybridisation to the part ofthe subsequent target polynucleotide and is revealed for hybridisationas the preceding target polynucleotide moves through the pore. 5.(canceled)
 6. A method according to claim 1, wherein a part of thesubsequent target polynucleotide selectively hybridises to a part of thepreceding polynucleotide and the preceding target polynucleotide isdouble stranded, wherein the part of the preceding target polynucleotideis in one strand and is hybridised to the other strand and wherein thepart is revealed for hybridisation as the two strands separate as thepreceding target polynucleotide moves through the pore.
 7. A methodaccording to claim 1, wherein the preceding target polynucleotide isdouble stranded and wherein both strands of the double strandedpreceding target polynucleotide are linked at one end by a hairpin loop,optionally wherein the other end of the preceding target polynucleotidecomprises a leader sequence which preferentially threads into the pore.8-9. (canceled)
 10. A method according to claim 1, wherein thesubsequent target polynucleotide is double stranded and the two strandsare linked at one end by a hairpin loop. 11-14. (canceled)
 15. A methodaccording to claim 1, wherein the one or more subsequent targetpolynucleotides are selectively attached to the first targetpolynucleotide by (i) covalent attachment; (ii) a ligase; (iii) atopoisomerase; or (iv) by click chemistry.
 16. A method according toclaim 1, wherein the movement of the concatenated polynucleotide throughthe pore is controlled by a molecular brake. 17-20. (canceled)
 21. Amethod according to claim 1, wherein the transmembrane pore is a proteinpore or a solid state pore. 22-26. (canceled)
 27. A method ofsequentially passing polynucleotides through a transmembrane pore, themethod comprising contacting a first target polynucleotide with the poreunder conditions in which the first target polynucleotide enters thepore, wherein, following its entry, an attachment site is revealed onthe first target polynucleotide, such that the first targetpolynucleotide attaches to a second target polynucleotide at theattachment site, thereby guiding sequential entry of the second targetpolynucleotide into the pore following passage of the first targetpolynucleotide through the pore.
 28. A polynucleotide adaptor comprisinga first polynucleotide strand and a second polynucleotide strand,wherein: (i) a portion extending to the 3′ end of the firstpolynucleotide strand is complementary to a portion extending to the 5′end of the second polynucleotide strand and the complementary portionsform a duplex; (ii) a portion extending to the 5′ end of the firstpolynucleotide strand and a portion extending to the 3′ end of thesecond polynucleotide strand do not hybridise to one another; and (iii)(a) the portion extending to the 5′ end of the second polynucleotidestrand comprises a sequence that is capable, when the duplex is unwound,of hybridising to a sequence comprised in the portion extending to the5′ end of the first polynucleotide strand; or (b) the portion extendingto the 5′ end of the second polynucleotide strand comprises a sequencethat is identical to a sequence comprised in the portion extending tothe 5′ end of the first polynucleotide strand.
 29. A population ofpolynucleotide adaptors comprising a first polynucleotide adaptor and asecond polynucleotide adaptor, wherein the first polynucleotide adaptorand the second polynucleotide adaptor each comprise a firstpolynucleotide strand and a second polynucleotide strand, wherein:(a)(i) a portion extending to the 3′ end of the first polynucleotidestrand is complementary to a portion extending to the 5′ end of thesecond polynucleotide strand and the complementary portions form aduplex; (ii) a portion extending to the 5′ end of the firstpolynucleotide strand and a portion extending to the 3′ end of thesecond polynucleotide strand do not hybridise to one another; andwherein (b) the portion extending to the 5′ end of the secondpolynucleotide strand of the first polynucleotide adaptor comprises asequence that is capable, when the duplex is unwound, of hybridising toa sequence comprised in the portion extending to the 5′ end of the firstpolynucleotide strand of the second polynucleotide adaptor.
 30. Apopulation of two or more polynucleotide Y adaptors for characterisingtwo or more double stranded target polynucleotides, wherein each adaptorcomprises first and second parts which are capable of hybridisingtogether and wherein each first part is initially protected fromhybridisaton to the second part.
 31. A population according to claim 30,wherein the first part is initially protected by hybridisaton to theopposite strand in the double stranded region of the Y adaptor.
 32. Apopulation according to claim 30, wherein one half of thenon-complementary region in each Y adaptor itself forms a loopstructure.
 33. A population according to claim 30, wherein one half ofthe non-complementary region in each Y adaptor itself forms a loopstructure and the first part is adjacent to, but not part of the loopstructure.
 34. A population according to claim 30, wherein each Yadaptor further comprises a leader sequence comprising the second part.35. A population according to claim 30, wherein each Y adaptor furthercomprises a leader sequence comprising the second part and wherein thesecond part is an overhang formed by a bridging polynucleotidehybridised to the free end of the leader sequence. 36-37. (canceled) 38.A kit for characterising two or more double stranded targetpolynucleotides, comprising a polynucleotide adaptor according to claim28 and one or more of the following: a population of hairpin loops; amicroparticle; one or more anchors capable of coupling a polynucleotideto a membrane; membrane components; and a magnet or electromagnet.